TW200930726A - Clottable concentrate of platelet growth factors and preparation method thereof - Google Patents

Abstract

The present invention relates to a clottable concentrate of platelet growth factors for therapeutic and/or cosmetic use, preferably comprising the growth factors PDGF, TGF-β , IGF, EGF, CTGF, bFGF and VEGF. In preferred embodiment said clottable concentrate of platelet growth factors does not induce blood cell-related transfusion reactions. The present invention also relates to a method for preparing a clottable concentrate of platelet growth factors according to the invention and comprising the steps of contacting a platelet concentrate with a solvent and/or a detergent, incubating said platelet concentrate with the solvent and/or detergent for a period of at least 5 minutes to 6 hours, at a pH maintained in a range from about 6. 0 to about 9. 0, and at a temperature within the range of from 2 DEG C to 50 DEG C, preferably within the range of from 25 DEG C to 45 DEG C, and removing the solvent and/or the detergent by oil extraction and/or chromatographic means.

Description

200930726 IX. INSTRUCTIONS OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The present invention relates to the field of platelet derivatives, and more particularly to the field of growth factor concentrates derived from platelets. The invention also relates to methods of preparing such growth factor concentrates, and to the use of clotting platelet growth factor concentrates in therapeutic and/or cosmetic applications. [Prior Art] The mechanism and path of directing wound healing and tissue regeneration have been studied in great detail, and it has been shown that most of the cellular and molecular event results after trauma are shared by different tissues in the body. Thus, the general mechanisms include early and late inflammatory response periods, cell growth and migration, angiogenesis, granulated tissue formation, and finally matrix formation and remodelling. Interestingly, these cascades are initiated immediately after the injury by the formation of blood clots, which are characterized by fibrin and various proteins that cross each other. The blood clots (vitronectin), fibronectin, and thrombospondin's blood clots prevent re-bleeding and serve as a substrate for invading cells while providing a barrier against pathogen invasion. In addition, this initial blood clot can be used to store the cellular derivatives needed in the later stages of healing. In particular, when we assume that all stages of tissue repair are centrally regulated by a number of factors, cytokines, and proteins, they are receptors that transduce and cross the cell membrane. Sexual interactions to adjust cell function. Later, the secondary message transducer controls the multicellular biology of intracellular 5 200930726. Although the roles and mechanisms involved in tissue regeneration are only partially “released”, most of the potential advantages have been confirmed, especially in the advantages of platelet derivatives, especially from gray platelets. Advantages of the platelet-derived growth factor. It is well known that platelet-derived growth factors exhibit chemotaxis and cell division properties and exhibit healing and regeneration with soft tissues and hard tissues (eg chemotaxis, cell proliferation, angiogenesis, cells). There is a direct correlation between extracellular matrix precipitation and remodeling) and enhanced cell proliferation. In addition, platelet derivatives are also indirectly related to some closely related biological functions, such as chemokine stimulated by bystander cells such as fibroblasts, macrophages, epithelial cells or lymphocytes. ) and the production of cytokines. Thus, platelet-rich formulations, as well as platelet gels, viscose and their eliminators, are used alone or in combination with transplanted biomaterials, and they serve as platelet derivatives in a variety of clinical or therapeutic applications. Or the source of platelet growth factor. 10 It has been found that such preparations containing platelet or platelet sources are particularly advantageous for the treatment of large bone injuries, complex cranioplasties and chronic wounds (eg oral and facial surgery, orthopedic surgery, periodontal disease). Surgery and plastic surgery): It is also particularly beneficial in the treatment of chronic ulcers, dental bone and soft tissue regeneration and oral implants; and in the treatment of musculoskeletal conditions and in the expansion of mesenchymal stem cells (by WVC) In terms of differentiation, and accelerating the proliferation of chondrocytes, endothelial cells and fibroblasts, platelet release is also subject to __ note, so its therapeutic advantages in cartilage re-treatment and gradual healing are also gradually affected by 200930726 local platelets. Source preparations (e.g., gels, gels, and elixirs) are typically prepared by mixing a platelet concentrate with an activator capable of eliciting release of the contents of the platelet particles to mimic the physiological activation of the platelets. The activation step is generally carried out by direct addition of exogenous thrombin or by the use of calcium chloride (CaCl2), which counteracts the effect of adding an anticoagulant during the collection of blood or platelets, triggering a blood agglutination chain reaction ( Coagulation cascade) 'and release endogenous thrombin. Endogenous and exogenous coagulase also induce fibrinogen (fibrin〇gen) polymerization and form fibrin-based biomaterials (blood clots), resulting in a variety of gray plate Multifaceted blends such as the release of various platelet growth factors. Recently, new research methods have been developed to produce recombinant platelet growth factors such as Regranex (human pDGF_bb) from conventional expression systems, and the synergistic effects of various growth factors can be observed. Therefore, compared with single recombinant factors, (Janssen Cilag Internat.). However, the available recombinant growth factors are still relatively small in number, in part because of the difficulty in isolation, identification, colonization and performance of these growth factors. Further, because

μ Ht- ra , ... tons of you, and the way of activation. Therefore, these variables obviously cause a significant difference in production (IV). The type of starting material, such as = degree storage; = material 7 200930726, and then the division of biological properties and clinical validity (such as healing power). For example, when a solution/gel containing intact platelets is delivered directly to the wound, the activation of platelets is associated with the rapid formation of fibrin clots, as well as the incomplete dissolution of platelets. Therefore, a considerable amount of intact platelets are still encased in fibrin hemagglutination, so it is difficult to determine the true release of growth factors.

Further, when the platelet gel and/or the growth factor preparation is obtained by the activation of thrombin and the excitation of the platelet, the fibrinogen is completely depleted when the gray small plate derivative is produced, The condensable state is therefore required to be mixed with exogenous hydrazine or synthetic product prior to delivery to the wound site. = "芡|詋, the therapeutic local platelet derivatives of the cerebral number are prepared from an autologous source. That is to say, the autologous platelet car is prepared by a few small or a few donated blood, and a fixed volume is used. The planned operation of the plate gel, solid. Disadvantages, because they are often in poor control, there is a lack of freshness to ensure that the growth factor (4) and its pre-J degree are ίί, In the case of strong factor thick liquid, it can be used to make plate growth agent, and it can be used for various special physiological and pathological conditions. The system is made up of (4) there will be the existence of #1 important deficiency, and thus in the blood of platelet source products. The immune response of cell debris to donor antigens. Acupuncture (4) Hair injection, allogeneic antigen immunity 200930726 f can be H丨if heavy consequences 'as observed in the transfusion reaction' and include the patient's autoimmune and dissolution Complications. J's current point is another major deficiency of existing platelet-derived products. 血小板 Platelet preparations derived from whole blood.) 2: Yes, but still when using platelet derivatives; ίίίϊ:ϊ = 染染. This allogeneic platelet formulations of platelet derivatives and gray small plate portion U "Finally, the increase in the aging population and chronic diseases will gradually grow became a health problem, which must be in the near future ^. Although there are a variety of therapeutic treatments available, including, for example, surgical sputum: birth = replantation, but for tissue wound healing and: after intensive research, the inventors were surprised to find that these targets = as described later The clotting platelet growth factor preparation method is achieved. The method can be simple, fast and effective in the preparation of gold platelets and organisms, and more particularly, can be obtained from individual blood], plate thick liquid or most people's small plate thick liquid (ροο1). Condensable blood small long-term factor thick liquid. More specifically, in comparison with the field of the related art, the method of the present invention will significantly increase the recovery of all the raw materials, at least the more important platelet growth factor, and 3 Others are the recovery of growth factors PDGF, TGF-β and EGF. Pay 9 200930726 c membrane virus and other pathogens:: The fat r is present in the initial platelet thick solution: .

Clinically, when platelet is used intravenously to treat Quantitative or Thromb〇cytopenia, due to blood, 丨=保二期 is 5 · 7 days (partly because of the risk of bacterial contamination), so A large number of platelet units with a storage time greater than 5 or 7 days are usually discarded in the mother year. In the case where an expired platelet stock solution is allowed to prepare a platelet-derived growth factor concentrate, the method of the present invention finally reveals a vision of a very economical goal. SUMMARY OF THE INVENTION One object of the present invention is a clotting platelet growth factor thick liquid' for use in therapeutic and/or cosmetic applications. In a preferred embodiment, the condensable platelet growth factor concentrate comprises growth factors PDGF, TGF-P, IGF, EGF, CTGF, bFGF and VEGF. In another preferred embodiment, the aforementioned clotting platelet growth factor concentrate does not induce blood cell related transfusion reactions.

In another preferred embodiment, the clotting platelet growth factor concentrate comprises at least one selected from the group consisting of fibronectin, transparent adhesion protein, thrombin sensitive protein, and von Willebrand factor. A protein of the group consisting of π, V, VII, VIII, IX, X, and XI 200930726 coagulation factors.另一 Another object of the present invention is a method for preparing a clotting long-factor thick liquid of the present invention, comprising the steps of: contacting blood, a thick liquid with a solvent and/or a detergent; and making the aforementioned platelets thick ^ with the aforementioned solvent and / or detergent maintained at a value from about 6 〇 to about 9 及 and at from 2. (: to a temperature within the range of 5 共同 to y 5 minutes to 6 hours, preferably at a temperature ranging from 25 to 45 C; and by oil extraction and/or chromatography To remove the aforementioned solvent and/or detergent. In the embodiment, the solvent or trialkyl phosphate used in the method of the present invention, the group consisting of two or three different alkyl chains And preferably a preferred embodiment of tri-n-butyl phosphate, a detergent for use in the method of the present invention, a polyoxyethylene derivative of fatty acid, and a partial vinegar of sorbitan S) , the group consisting of secretive _, deoxycholic acid and continuation (-s); and better ί 45, Trit〇n χ-ι〇〇 and ―0 are composed of hearts; Preferably, the final concentration of the above-mentioned dissolved SH cleansing side of the method of the present invention ranges from g. 2 to 5 ίί' ΛΙ 2 to 2% by volume, which is calculated based on the aforementioned initial platelet. A preferred embodiment of the method of the invention is 1% Triton X_45 contact, which is calculated based on the volume of the aforementioned plate/agricultural thick liquor. In a preferred embodiment of the invention, the oil extraction is carried out by a doctor, and the aforementioned oil is used in an amount of from 2 to 2 2009, 200930726 or from 5 to 15% by weight, or from 5 to 1% by weight, It is calculated based on the amount of the above-mentioned platelet concentrate, the mixture of the liquid and the solvent and/or detergent. In a preferred embodiment, the chromatographic method uses, for example, an 18-emulsified ruthenium filling material. Or SDR (solvent_detergent removal, o: t-Detergent removal) hyperD to remove the pre-cleaning agent. Each 鸮1 illusion is a preferred embodiment, the method of the present invention further ❹ 所 g Up to 75-nm, a filter or similar virus i, in addition to the spinel platelet growth factor thick solution, in a preferred embodiment, the method of the present invention comprises an initial platelet thick solution In a preliminary step, the aforementioned starting platelets are prepared from blood by platelet separation or buffy-coat separation, and are in a state of being fresh or expired and stored in a liquid state, and stored in a reduced state. / Invented another - the purpose is to form a blood coagulation The method of blocking, the bean line or the platelet growth C / chen thick liquid obtained by the method of the present invention is mixed with thrombin. Preferably, the clotting method used in the method of forming a blood clot is obtained from Further, in the second embodiment, the activity of the U to i volume, = the condensable blood of the square of the invention, is another object of the invention being a pharmaceutical product or artificially comprising the invention or by the invention The condensable 'blood f-plate growth factor thick liquid obtained by the method. 12 200930726 Another object of the present invention is to use the present invention or the use of the coagulated platelet growth factor thick liquid obtained by the present invention to form blood coagulation Block, or for cell regeneration or wound healing, in vitro study (kw plus) or in vivo study (exvivo) of cell pull = live [embodiment] One of the objects of the present invention is a clotting platelet thick concentrate 'It is used for therapeutic and/or cosmetic purposes. Factor Ο Ο The term "coagulation" as used in the present invention refers to a platelet growth factor concentrate, moon or fiber egg obtained by the method of the present invention. Original (flbrin〇gen) and a second coagulation factor, when the message contains a therapeutic applications, it may be with a suitable activating agent (e.g., when the helmet so that there is generated by mixing blood clot. To turn)

In a particular embodiment, the concentration of fibrinogen in the condensable long-factor thick liquid of the present invention is preferably higher than that of the small plate thick liquid, and more preferably higher than 1.5 g/L thick liquid. Preferably, it is higher than g/L thick thick liquid; and its concentration of factor XIII is preferably higher than 〇5 '.5g/L thick liquid, more preferably higher than 〇.75 IU/ml thick liquid, and more preferably thick IU/ml thick solution. (10) 1.0 1.0 The term “cosmetic use” as used in this article refers to the treatment of various body parts of the human body, especially skin, hair, nails, lips, external reproductive organs, or with skin and skin. The membrane is in contact with it to make it clean and fragrant, and to protect and keep the face in good condition. The role of the dual, the therapeutic use of the therapeutic use of the invention, or the therapeutic or prophylactic treatment of the human, and/or the motility, the disease, the medicinal, the immune or the metabolic effect Silk rejuvenation, 13 200930726 changed its physiological function. The condensable platelet growth factor concentrate obtained by the present invention or by the method of the present invention is particularly suitable for treating human and/or animal conditions, and/or for contact with body parts of the body because it has been removed Both solvent and detergent, and are viral safe. The term "clearing solvent and/or detergent" means that the amount of solvent and/or cleaning agent in the condensable blood small-plate growth factor thick solution is extremely low, and is preferable. Indeed, it is known to those of ordinary skill in the art that elevated concentrations of solvents and/or detergents are directly linked to long-term toxicity and, in particular, directly linked to the development of neurological disorders (as disclosed in JPR Pelletier, S · Transue, EL Snyder. Pathogen inactivation techniques. Best Practice & Research Clinical

Haematology Vol. 19, No. 1, pp. 205-242, 2006). Therefore, the term "clearing solvent" in the present invention means that the amount of the solvent is less than 100 ppm', preferably less than 50 ppm, more preferably less than 20 ppm, still more preferably less than 10 ppm, less than 5 ppm, and even better. Further, in the present invention, the term "cleaning agent" means that the amount of the cleaning agent is less than 500 ppm, preferably less than 250 ppm, more preferably less than 100 ppm, and even more preferably less than 50. Ppm, and even more preferably small at 10 ppm. "Virus safety" means that the clotting platelet growth factor thick liquid is substantially free of infectious virus, especially without lipid-containing viruses, such as human immunodeficiency virus, hepatitis B virus. (HBV), hepatitis C virus (HCV), West Nile virus (WNV), TT virus, dengue virus, cytomegalovirus (CMV),

Epstein Barr virus (EBV), human herpesvirus-8 (HHV-8), 200930726 simian foam virus, severe acute respiratory syndrome virus (SARS coronavirus) and other lipid envelope hepatitis viruses, cytomegalovirus, milky scorpion Enzymatic disease, Herpes group virus, rhabdovirus, leuk〇virus, mucus virus, alphavirus, arbvirus, paramyxovirus, squamous virus (arenavirus) and coronavirus. “Substantially without” means that the degree of virus deactivation of the clotting platelet growth factor thick solution is at least greater than 4 logl ◦, which is the standard set by the powerful virus attenuating step (by ❹

The European Agency for the Evaluation of Medicinal Products (EMEA), the Committee for the Evaluation of Medicinal Products (CPMP), is based on the guidelines for the approval of viruses (N〇te f〇r guidance on vims validation studies). ), the reference CPMP/BWP/268/95) 'and more preferably greater than 5 l〇g1G or further greater than 6 log1(), so 'it is unlikely to cause a lipid envelope virus when transfused to a patient Blood-borne infection. The clotting platelet growth factor concentrate obtained by the present invention or by the method of the present invention further comprises the following functional growth factor: gold platelet growth factor (PDGF), which is a heterodimer of A and B chains and/or a homodimer of AA and/or BB chain (PDGF-AA, PDGF^ve, PDGF-BB); a superfamily of transforming growth factor (TGF-β), which specifically comprises TGF-βΙ and/or TGF-P2 and / or bone morphogenetic protein ^b〇ne morphogenetic protein, BMP); insulin-like growth factor (IGF); epidermal growth factor (EGF); connective tissue growth factor (CTGF); primary fibroblast growth factor (bFGF) and gold tube Endothelial growth factor (VEGF). In a special embodiment, the clotting platelet of the present invention 15 200930726 long factor, the concentration of PDGF in the thick liquid is higher than 9 ng/mUf thick liquid, preferably higher than 1 ng/ml thick. Preferably, the liquid is more than 12 ng/ml thick liquid, more preferably higher than 150 ng/mi thick liquid, more preferably higher than 180 ng/mi thick liquid, and even more preferably higher than 25 〇 tons such as 丨 thick liquid. ❹ ❿ In a special embodiment, the concentration of TGF-βΙ in the condensable platelet growth factor thick solution of the present invention is higher than 1 ng/ml thick liquid 'preferably higher than 140 ng/ml thick liquid More preferably, it is higher than 16 ng/ml thick liquid, and more preferably higher than 18 ng/mi thick liquid, and then 250 ng/ml thick liquid. In a special embodiment, the concentration of IGF in the condensable blood small factor thick solution of the present invention is at least the same as that of the starting platelets, and preferably more than 65 ng/ml thick solution, and more 75 ^-Thick liquid 'is better than the thick liquid of the heart secret is better than 100 ng / ml thick liquid. In a special embodiment, the # EGF concentration in the condensable long-factor concentrated liquid of the present invention is higher than 〇5 η_concentration, and is 11,1 thick liquid, more preferably higher than 1.5 ng/ The heart palpital solution is more preferably a 2 ng/ml thick solution and a ng/ml thick solution. In order to ask 2.5 in the special implementation of neot, the present invention is a platelet growth factor == platelet growth factor indifference liquid is suitable for use = "requires low lipid content" as used in the present invention or refers to the method of the present invention Growth factor thick liquid system has been removed fat f (deplet ^ blood is small, that is, in this (four) material R ^ 16 200930726 = raw $ factor indifference liquid t, cholesterol, triglyceride, vinegar, dish and the day after the day is low a lipid bipolar in a concentrated platelet-derived growth factor thick solution in a concentrated growth factor thick solution known as the starting platelet thick solution=Γΐ日=, _chemical field (especially cholesterol, triglyceride and LDL) The amount is extremely low. 2 The Γ 液 liquid is suitable for more therapeutic use, for example, for patients with high risk of cardiac complications, or for high risk of heart disease. Patients treated are extremely low, and the risk of vascular occlusion caused by the formation of plaques in the arteries and venous systems is greatly reduced. Conclusion ================================================================ r; & surname Λ thick, the amount of cholesterol is less than 100 can be condensed: blood: plate t' is preferably less than 5 〇 heart _ condensable and more preferably lower than the knot ί = raw hair:: method of condensable 100 mg / dl condensable q, plate medium length due to acid The amount of glycerol ester is less than 5 可 condensable ▲ small plate growth factor concentration ^, plate cleavage ^ 枝 麟 的 的 的 mg mg mg mg / / / mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg H H H H H H The amount of coffee is less than 30 mg = the amount of coagulated platelet growth factor concentrate is lower than the present invention or # condensable gold plate growth factor thick solution obtained by the method of the present invention, the amount of coffee is lower than = 17 200930726 mg/dl clotting platelet growth factor thick solution, preferably less than mg/dl clotting platelet growth factor thick solution, more preferably less than 2 〇mg/dl clotting platelet growth factor thick solution, and Still more preferably less than 5 mg/dl of clotting platelet growth factor concentrate. Further, the quality of the lipid in the clotting platelet growth factor concentrate obtained by the method of the invention or by the method of the invention is very low and/or • Undetectable, so there is better stability, when large-scale purification of platelet growth factor The method is relatively easy to perform. φ In a special embodiment, the condensable platelet growth factor thick solution obtained by the present invention or by the method of the present invention does not cause blood cell-related transfusion reaction. "This clotting growth factor concentrate does not contain intact living cells (such as red blood cells, platelets, and white blood cells), so it can avoid causing a range of known transfusion reactions in patients, including immunity (such as allogeneic immunity (" Alloimmunization) and hemolytic complications (see, for example, Str〇ncek et Rebulla, «Platelet transfusions», The Lancet, August 4 2007; vol, 370: 427-438). Indeed, recipients with normal immune abilities typically exhibit an immune response to the donor's blood cell antigens, resulting in a variety of clinical outcomes depending on the relevant blood cells and specific antigens. The most common related antigens are selected from the following categories: (1) type 1 • HLA, which is a combination of platelets and white blood cells; (2) type 2 HLA, which is expressed in some white blood cells; (3) granulocyte-specific An antigen; (4) a platelet-specific antigen (for example, a human platelet antigen HpA); and (5) a red blood cell-specific antigen. More specifically, in the preparation method of the present invention, living blood cells (red blood cells, white blood cells, and platelets) are destroyed/lysed by solvent detergents to reduce (and more preferably prevent) patients from exposure to foreign antigens.

200930726 ΐΪίίί Cell risk. Therefore, the method of the present invention can be made from a small plate prepared by a patient (for reading the patient itself), or from the blood of the allogeneic J plate: a clotting platelet growth factor thick solution. 2. Specially implemented secret towel, the present invention or the method of the present invention has two kinds of selective ΓΓ platelet growth factor thick liquid into the step consisting of ί egg 22 = white X: XI coagulation factor and Van der Boer factor Further, in a special embodiment, the concentration of fibronectin in the clotting factor of the platelet growth factor of the present invention is preferably higher than that of the 〇2 g/L thick solution, and more preferably about 〇3. g/L thick liquid. In the secret of the present invention, the clotting factor of the 11th, IX, X, VII and/or XI in the condensable, plate source growth factor thick solution of the present invention is preferably higher than 0.51 wrist 1 thick liquid' More preferably, in the special implementation, the individual concentration of the vm coagulation factor and/or the van der Waals factor in the condensable platelet growth factor thick solution of the present invention is preferably higher than 0.5 iu/mi thick liquid. More preferably, it is an ancient sputum. 9· thick liquid, and more preferably... two: for-in another embodiment, the condensable platelet growth factor thick liquid obtained by the method of the present invention further comprises an immunoglobulin Proteins such as IgG, IgM and/or IgA. In a specific embodiment, the concentration of the immunoglobulin from the IgG 可 in the condensable platelet growth factor thick solution of the present invention is preferably higher than 5 g/L; and the thick liquid solution is more preferably about 1 〇g/L. Thick liquid. In another embodiment, the condensable platelet growth factor concentrate obtained by the present invention or by the method of the present invention 19 200930726 further comprises at least one growth factor selected from the group consisting of: CCN family members Connective tissue activating protein-3 (CTAP-3), PF4, platelet-derived angiogenic factor (PDAF), endothelial cell growth inhibitor, early pregnancy factor (EPF), epidermal growth inhibitor (EGI), keratin Cell growth factor (KGF), angiopoietin-like protein _6 (ANGPTL6), IGFBP-3, estrogen receptor-associated protein, - fibroblast-derived endothelial cell growth factor (f-ECGF), hepatocyte growth factor (HGF), histamine release factor, human collagenase inhibitor, platelet anti-protein-1 (PMP-1), thrombin-induced platelet antibacterial protein-1 (1>1>1^0>), coagulation Peptide_1 (such as 〇11115〇(^11-1,1^1) and thrombincidin-2 (TC2). In another embodiment, the invention is exemplified by the method of the invention Suspected platelet growth factor thick solution Further comprising at least one protein selected from the group consisting of serotonin, tissue protein S# (cathepsin), albumin, platelet facial protein _pjgp (CXCL7), neutrophil activating protein-2 and _4 (NAp_2, _4), somatostatin (SST), RANTES, CTAP-3, placental protein 14 (PP14), SCUBE annexin n (annexinll), heat shock protein 27 (HSP27) And heat shock protein 60 (HSP60) - In a particular embodiment, the concentration of albumin in the clotting platelet growth factor concentrate of the present invention is preferably greater than 30 g/L of concentrated liquid. In another embodiment, the condensable platelet growth factor concentrate of the present invention or the method of the present invention further comprises at least one enzyme selected from the group consisting of collagenase, superoxide Superoxide dismutase (SOD), heparinase, 20 200930726 metalloproteinases MMP-1, -2, -9, -13, autocrine ERK (ext.Cell reg. kinase), autocrine and paracrine Protein C (PC), and trace amounts of enzymes such as aldolase Defensin, acid structase, arylsulphatase, β-galactosidase, β-glucose acetoin, β-glycerolphosphatase, α/ β·glucoine enzyme, α/β-fucosylase (α/β-fucosidase), oc-mannose's glycosidase (α-mannosidase) and α_arabinosidase. (α-arabinosidase). 0 In another embodiment, the condensable platelet growth factor concentrate obtained by the present invention or by the method of the present invention further comprises histamine, ADAMTS-13, al-oc2 antitrypsin, α2_anti-cytosolic ( A2-antiplasmin), α2-macroglobulin, ci-INH, induced sputum-2, -6, -7 (TGF-β superfamily), ECM remodeling factor (ECM remodelling factor), induced sputum, TNF- α, elastase......), autocrine and para; lysophosphatidic acid (LPA), HMGB1 (amphiregulin), ATP, ADP, GPT, GDP, Ca2+ , Mg2+ and/or Zn2+. Another object of the present invention is a pharmaceutical product comprising the condensable platelet growth factor • concentrated solution of the present invention or obtained by the method of the present invention. - "Pharmaceutical product" means a platelet gel, a platelet glue, a growth factor-rich fibrin glue and/or a sealant, an artificial eagle. Another object of the present invention is the use of the coagulated platelet growth factor of the present invention or the method of the present invention in a medium, wherein the medium is suitable for use in fibroblasts, chondrocytes, osteoblasts, keratinocytes, stem cells And/or transplantation of cells in vitro or in vivo. In the present invention, another factor, a cell, a chondrocyte, a nucleus, or a horn of the nucleus fiber # invention or the method of the present invention. Platelet growth of r嘁 knot Another object of the present invention is to grow a small plate of gold called a thick layer (four) method ϋ I == condensed 1 start platelet thick liquid and _ and / or Qing 3 from 2 small plate thick liquid Contact with the solvent and / / 戋 clear, so that the initial blood is about 9 ·. Within the range = and at about 6. To co-culture at least 5 minutes to 6 degrees C to 45 ° C, Tian Sheng Push and · TU Wen Jia is to remove the aforementioned solution or clear by oil extraction and / or Layers to 4:; pH values in the range of ==, = ΓΛ8 to 8.2 (when the initial blood is small or plum plate). Advantageously, the culture enthalpy-(η-hexyl) acid vinegar, tris-(2-ethylhexyl) oleate, 1 ^ hexanol, such as butyl, used in the process of the invention. She 1, 2 ♦ butyl) phosphorus ', 3

St. Burning base chain of the second burning class _ class: = = soil one or alkyl phosphates, such as bis-(η-butyl) phosphonium phthalate is preferably dialkyl phosphate is tri-(η-丁Phosphate 22 200930726 (ΤηΒΡ) 合适 Suitable cleaning agents for use in the process of the invention include polyoxyethylene derivatives of fatty acids, partial esters of sorbitan (for example, the product name Tween 80) (also known as "Polysorbate 80" ("polysorbate 80"), "Tween 20", and non-ionic detergents (such as oxyethylated alkylphenols sold under the following product names: "Trit〇n Χ_ι〇〇” or “Triton X-45”). Further considerations for the detergents are sodium deoxycholate and sulfobetaines such as N-dodedo-n,N-didecyl-2-ammonium-1-ethanephthalate (N-dodecyl) -N,N-dimethyl-2.-ammonio-l-ethane sulphonate ). The best cleaning agents are "Triton X-45", "Triton X-100" and "Tween 80". In a particular embodiment of the invention, the initial gold plate thick liquid is co-cultured with a solvent and a detergent, preferably co-cultured with ΤηΒΡ and Triton X-45. Advantageously, the final concentration of the aforementioned solvent or the aforementioned cleaning agent, or the respective final concentration of the aforementioned solvent and the aforementioned cleaning agent is included in the range of from 0.2 to 5% by volume, preferably contained in from 0.2 to 2% by volume. Within the range of 'based on the volume of the aforementioned platelet thick liquid. In a preferred embodiment, the aforementioned initial platelet rich liquid system is co-cultured with 2% ΤηΒΡ. In another preferred embodiment, the aforementioned initial • gold platelet thick liquor is co-cultured with 1% ΤηΒΡ and 1% Triton Χ-45. The solvent and/or detergent may be extracted from the biological fluid by oil extraction with a pharmaceutical grade oil and/or other methods such as column chromatography, such that the solvent and/or detergent in the thick liquid Has been removed. The aforementioned pharmaceutical grade oil may be a natural oil (e.g., an oil extracted from plants or animals) or a structurally similar synthetic compound. Suitable natural 23

200930726 Oil includes castor oil (castor oU, or ridnus 〇u), soybean oil, sunflower oil, cottonseed oil β. The preferred synthetic compound is synthetic triglyceride. Examples of suitable synthetic triglycerides include triolein, tristeadn, tnpdmitin, triterpenoid (f), and combinations thereof. The amount of the medical grade oil is extractable at least 8% by weight of the fat-soluble process chemical, (process chemica!), the amount of oil used is from 2 to 2 〇 ff% 'the fine lysate of the plate thick liquid The basis weight is from 5 to 15% by weight, and more preferably from 5 to ι by weight. The oil extraction can be carried out once, preferably twice, and more preferably three times, depending in particular on the concentration of the oil. The aforementioned solvent and/or cleaning agent can also be removed from the smear of the thick plate by means of column chromatography. Tube (iv) analysis can also be carried out after oil extraction or directly after solvent and/or detergent treatment. Suitable chromatographic columns available include reversed phase (hydrophobic interaction) tannins, or protein-adsorbing matrices such as ion exchange (anionic and cationic) matrices and affinity (eg, immuno-affinity or immobilized heparin) matrix or ruler = exclusion ( Size-exclusion) matrix. Preferably, the reverse phase substrate is ci8: emulsified Shishi loading material, SDR (solvent__cleaner removal) such as Ke D 0> aU c〇rporation), polystyrene absorbent (ν & Γί^ ) and

Amberlyte (Rohm). Even if the c丄8 series is generally used for the better choice of industrial scale chromatography, it will be considered as a dioxo-filling (four). These adsorbing substances are used in combination with the aforementioned growth factor when the aforementioned solvent and detergent are washed out in the leading fraction. The anion exchange matrix depends on the growth factor to be purified, preferably an anion exchange gel such as DEAE_S叩hadex A-50. DEAE-Sepharose FF > Q-Sepharose ^ DEAE- 24 200930726

Toyopearl 650M, DEAE-Hyper D. These adsorbing substances are used for the binding of the growth factor when the aforementioned solvent and detergent are eluted in the leading edge portion. / In a preferred embodiment, the amount of solvent after oil extraction and/or chromatography is less than 100 ppm 'preferably less than 50 ppm', more preferably less than & ppm' and even more preferably less than 1 〇ppm' Less than 5 ppm, and even more preferably less than 1 ppm.

In a preferred embodiment, the cleaning dose after oil extraction and/or chromatography is less than 500 ppm', preferably less than 250 ppm, more preferably less than 100 ppm', and even more preferably less than 50 ppm, and further More preferably less than ppm. After removing the solvent and/or detergent by oil extraction and/or chromatography, a further step can be added to make the non-enveloped virus (such as small/ill B19, or possibly Is Hepatitis A Virus (HAV)) deactivated or: removed, such as nanofiltration' and more particularly using 75_nm, 35_nm, 20-nm, 15-nm or 10-inch pore size membranes for nanoparticles Too much. In another preferred embodiment, the centrifugation step is performed on oil extraction and/or chromatography to remove cell debris. Advantageously, the foregoing is carried out at 800 to 20000 X g for 1 to 3 minutes and for 15 minutes for 10,000 X. Horses In another mode of treatment, the oil extraction and/or chromatography is followed by a filtration removal step to remove cell debris. Advantageously, the pre-filtration step is performed with a filter that varies gradually from 1 μm to 0.2 μm, which also removes bacteria. ~ In a special embodiment, in the method of the present invention, the platelet thick liquid of the material is single or mixed: a small plate thick liquid, for example, a platelet thickening for use in the production of ▲. Enterprise 25 200930726 Small plate thick liquid can also be obtained from the whole blood buffy brown layer separation method. A unit of platelet thick liquid from the blood sedimentation layer is generally 3 to 5 〇 mi. Platelets obtained from jit > granules can be used as a starting material, which is a single unit or a mixture of a plurality of single units, such as a mixture of 4 to 6 single units when forming a therapeutic unit ( For example, Guide t〇the扒叩虹此,, and use assurance of blood components _ 13th edition (2007), edited by the Council of Europe. Gold plate thickening can be performed by jk liquid separation (referred to as platelet preparation by whole blood donation of φ), cell separation (cytapheresis) or plateletpheresis standard procedure (see, for example, Guideto the preparation, use and quality Assurance of blood components, 13th edition (2007), edited by the Council of Europe) is derived and can be derived by using MCS+ (Haemonetics), Trima Accell or COBE Spectra (Gambro) or Amicus (Baxter). In contrast to those obtained through the buffy coat separation procedure, platelet separation typically results in a larger volume (equivalent to 300 ml of platelet concentrate) from each donor. In a preferred embodiment, the method of the present invention comprises a preliminary step of preparing a starting platelet cullet. Advantageously, methods for preparing a starting platelet concentrate include, but are not limited to, standard blood bank procedures (eg, small blood, plate separation, or platelet preparation via whole blood donation) and priority care procedures (eg, using blood cell preservatives/ Separator or table device). The platelet concentrate used as a starting material in the method of the invention may be fresh (ie less than 5 or 7 days after collection), expired (ie more than 5 or 7 days after collection), or expired and frozen at _2 Hey. (: or the following weeks. In a particular embodiment, the platelet concentrate used as the starting material 26 200930726 in the method of the present invention may further comprise white blood cells and/or red blood cells. Thick liquids may contain several differentiated and unactivated white blood cells, such as lymphocytes, neutrophilic granulocytes, and single and globules. More specifically, neutrophils and mononuclear spheres are rich in bone marrow peroxidation. a particle of myeloperoxidase, which catalyzes the oxidation of chlorides to produce hypochlorous acid and other reactive oxygen derivatives, wherein the reactive oxygen derivative is used as a bactericidal oxidant, Microorganisms and molds are toxic. ❹ Therefore, when the starting platelet concentrate contains several differentiated and unactivated white blood cells, the condensable platelet growth factor thick solution obtained by the method of the present invention further comprises an antimicrobial derived from white blood cells. In a preferred embodiment, the white blood cells in the platelet concentrate used as the starting material are destroyed, preferably by Leukoreduction is used to 'avoid the pro-inflammatory response of proteases and acid hydrolases in white blood cells. Leukocyte depletion also reduces the risk of prion contamination. 〇 Normal human platelet counts are generally The mm3 blood contains 150,000 to 400,000 gold platelets, which is 150 to 400 X 1 〇 9 platelets/L. This "normal" platelet count is about 95% healthy people's, and the remaining 5% may There will be statistically abnormal platelet counts (very low or very high). When platelets are collected by platelet separation, the platelet count in a bag of 250 ml is generally higher than 1.2 X 1 〇 9 platelets per ml. The platelet count is equivalent to more than about 3 χ 1 〇 " platelets per bag (unit). In the preferred embodiment, 'the blood in the initial gray plate thick liquid 27 200930726 small plate number ^ is better than The number generally seen in ash is 3 to 1 times higher. Another aspect of the invention is a method of forming a blood clot which is carried out by the following steps: the invention or by the method of the invention The resulting clotting platelet growth factor concentrate is mixed with thrombin or with another activator of blood agglutination, such as activated VII or bovine or human thromboplastin. Or the condensable platelet growth factor concentrate obtained by the method of the present invention or the anti-fiber may be added to the condensable platelet growth factor concentrate obtained by the method of the present invention or the blood clot formed by the method of the present invention. A protein solubilizing agent to inhibit or slow down fibrin/glutamate, a natural proteolytic enzyme (such as plasmid) that breaks down fibrin clots. In a particular embodiment, the anti-fibrinolytic agent is aprotinin 'but concentration> 1 〇 mg/ml of tranexamic acid (additional acid - acid) or ε-aminohexanoic acid ( Epsiil〇namin〇capr〇icaeid) can also be used as an alternative compound for ruthenium peptides. In the liquid form, the concentration of the inhibitor peptide is usually 3000 KIU or less. After mixing the clotable platelet ❹ growth factor concentrate and thrombin components, the final concentration of the pancreatic peptide is typically half that of the starting solution. The condensable blood small, plate growth factor thick solution obtained by the method of the present invention or the method of the present invention can be further mixed with an artificial scaffold (for example, a manufacturer of collagen, chitosan, ceramics), or A mixture of plasma fibrin glue or fibrin sealant. Additional compounds may also be added to the condensable platelet growth factor concentrate of the present invention prior to mixing with thrombin. Such additional components include, but are not limited to, chemotherapeutic agents, antibiotics, and/or hormones. Mixing the condensable, such as 28 200930726 small plate growth factor thick solution obtained by the method of the present invention with thrombin, reproduces the final step of the blood coagulation chain reaction, and gradually polymerizes fibrinogen to form Blood clots or insoluble fibrin. The term "thrombin" as used in the present invention relates to thrombin which can be obtained from any source, and preferably refers to bovine thrombin; if it is used for human therapeutic applications, it is more preferably human coagulase . ❹ amp & (^12 activated human plasma or python thrombin (batroxobin, another fibrinogen coagulation protein ' ' from the snake ^ ^ 仏 (7) ^ plus corpse (8) public (9) Toxic), activated FVII, or human or bovine thromboplastin can also be used as a replacement for thrombin. In a preferred embodiment, 1 to 1 volume of thrombin is added to 1 volume. The invention or the condensable ▲ small plate growth factor thick solution obtained by the method of the invention. In a preferred embodiment, the thrombin concentration range is from 20 IU/ml to 1000 IU/m. , the final concentration of thrombin is about 500 IU / m to ensure that continuous and rapid fibrinogen polymerization will first obtain soluble fibrin, and then obtain stable (insoluble) Ο fibrin; or when In the case of special surgery, in order to slow down the polymerization, the final concentration will be lower, about 4 to 25 IU/ml. Two components (ie, the present invention or the clotting platelet growth obtained by the method of the present invention) Both factor thick and thrombin) can be double The cartridge system is applied sequentially or simultaneously at the location to be repaired 'with or without the use of an endoscope delivery device, and is sprayed or easily absorbed (see Rad sevich et al. , Fibrin sealant · Scientific Rationale, Production Methods, Properties, and Current Clinical use55, Vox Sanguinis, 1997, 72:133-143 and Marx G, "Evolution of fibrin glue 29 200930726 applicators", Transfus Med Rev, 2003; 17 (4 The application method described in 287-98 is incorporated herein by reference. Therefore, another object of the present invention is the therapeutic use of the condensable platelet growth factor thick solution obtained by the present invention or by the method of the present invention. Uses, and uses for forming blood clots, or cell culture applications for in vitro or in vivo studies. When used for cell culture in vitro or in vivo studies, the present invention or the coagulation obtained by the method of the present invention • The platelet growth factor thick solution will appear in the medium in an amount from 1% to 30%, preferably from 2% to 20%, and Also more rabbits 舛 3 ° /. to 10%, based on the volume of the medium. The main therapeutic applications of the aforementioned thick liquid / thrombin mixture include but not limited to: dental surgery, implantation, orthopedics and oral surgery , surgery, soft tissue and hard tissue healing and reconstruction, cardiovascular, thoracic, or monthly bowel surgery, neurosurgery, general surgery / trauma surgery, ophthalmology, otolaryngology, urology, and in patients with anticoagulation Or surgery on a patient with poor coagulopathy. The above and other objects, features, and advantages of the present invention will become more apparent from the description and appended claims. Pharmacology I. Materials and Methods 1.1 - Separation of the akes platelet The initial platelet concentrate (PC) was obtained using the MCS ten-component system (Haemonetics, Braintree, USA) with the consent of the volunteer donor. Collected. Whole blood is obtained through an intravenous catheter using intermittent flow and anticoagulant (1 ml anticoagulant citrate dextrose solution formulation per 10 ml of blood). Platelet-rich plasma 30 200930726 (PRP) is automatically separated from other blood components by centrifugation and collected in a sterile, single-use disposable bag, and red blood cells and plasma are returned to the donor. This cycle is repeated until a predetermined volume of PRP (about 300 ml) is obtained. The starting platelet thick liquor was processed within 24 hours of collection as described below. 1.2- Blood cell count Platelets, white balls (WBC) and red flag counts were measured using a cell q counter (ABC Vet Automatic Blood Counter, ABX Diagnostics, France). 1.3- Treatment of the starting platelet concentrate a-Study design The initial platelet thick solution was processed according to the study design of the first figure. Briefly' The same donor starting platelet concentrate (3 〇〇 ml) was gently mixed' and divided into two equal volumes of pool (150 ml). Subset Pool 1 is directly subjected to S/D processing' without prior activation. The subset pool 2 is in the presence of ❹ 23 mM CaCl 2 and glass beads (beads), which will make the gold small

The plate gel is activated under conditions of formation, as described below; the resulting release is carefully recovered by pipetting (average volume is equivalent to 12 〇-m b because there is a loss of 30 ml in the small plate gel Medium), then subjected to S/D treatment followed by oil extraction. Among the samples obtained from the starting platelet thick solution, there are S/D-treated unactivated gray plate subset pools, platelet-activated subset pool 2, and S/D-treated activated subsets. Pool 2. 200930726 b-jhi platelet activation: In the presence of glass pellets, 1 M CaCl2 (Sigma, lot 056k0688) was added to the jii platelet concentrate (subset pool 2) for activation to a final concentration of 23 mM. . The mixture is gently spun and mixed until a blood clot forms, which typically takes place in 5 to 8 minutes. The mixture was allowed to reactivate for a period of 60 minutes, at which time the optimal platelet-derived growth factor was released under these experimental conditions. • The supernatant is poured out and separated from the glass pellet/formed fibrin clot. The average supernatant recovered was about 80% of the starting platelet concentrate. The supernatant will be further processed by s/d. c-S/D treatment: For the sake of convenience, the S/D treatment of the unactivated subset pool 1 and the activated subset pool 2 is carried out in a bag as described in EP 1 685 852. Briefly, a 50%/50% mixture of TnBP (Merck KGaA, Darmstadt, Germany) and Triton X-45 (Sigma, Missouri, USA.) was added to each subset pool for 15 minutes and continued to mix to maximize The final concentration of φ (v/v) was 1% TnBP and 1% Triton X-45. After complete addition: The mixture of S/D-platelet subset pools was shaken vigorously for 1 minute. The treatment bag was then completely immersed in a water bath and the S/D platelet mixture was warmed to 25 ± 0.5 ° C' and then treated for 1 hour with continued mild disturbance. Upon completion of the S/D treatment, soybean oil (Sigma, Missouri, USA) was added to the S/D-platelet subset pool to a final concentration of 10% (v/v). The bag was shaken vigorously for 1 minute and then placed in a rotary shaker for 15 minutes. The platelet subset pool (lower layer) was removed from the oil (upper layer) in a pour-out manner (20 minutes), and transferred to the second bag by gravity, and the oil extraction was repeated three times. This oil extraction procedure reduces TnBP and Triton 32 200930726 X_45 to less than 10 and 100 ppm, respectively. 1.4- Effects of centrifugation and lysing agent types In order to study the effects of platelet content and S/D reagent types on the release of platelet-derived growth factors, an experiment was conducted: dividing platelet concentrate (300 ml) into two i5 〇ml subset pool. One of the subsets was centrifuged at high speed (10000 X g) to make the platelets a pellet and the supernatant was treated with ι % ❹ TnBP-l% Triton X-45. Another uncentrifuged subset pool was subdivided into two 75 ml subset pools, one of which received 1% TnBP-1% Triton X-45 treatment and the other received 2% TnBp treatment. S/D treatment (culture and oil extraction) was carried out as described above. 1.5- Bovine Thrombin Activation Assay Assuming that CaCl2 activation does not completely activate platelets, the two platelet concentrates (H ml) were activated as described above in the presence of 0.23 M CaCl2 and glass spherules. G After gently mixing for 60 minutes at room temperature, 10 ml of platelet thick liquor release was recovered, and 0.5 ml of 1000 IU/ml bovine thrombin (Thrombin-JMI, 52604-7102-1, was added. Jones • Pharma, Saint_Louis, MO) to a final concentration of approximately 48 IU/ml. The mixture was gently mixed and spun at room temperature for 60 minutes. After that, 1% TnBP-1% Triton X-45 treatment and oil extraction were performed. In a parallel experiment, two platelet concentrate samples (10 ml) were also directly activated with 0.5 ml of the same bovine thrombin, gelled in a few seconds, and then gently mixed for 60 minutes. Samples were taken in various steps of the experimental method, centrifuged at 10,000 xg and frozen at -80 °C until blood platelet growth analysis was performed. 1.6-Growth Factor Determination The 丨-(6) sample was taken at each step of the procedure. Centrifuge at 1 〇〇〇〇 X g for 15 minutes (Microfuge® 22R, Beckman Coulter,

Fullerton, CA) 'A small plate and/or cell debris is made into a mass, and a cell-free supernatant is obtained for platelet-derived growth factor measurement. In addition, a parallel experiment of 15 minutes was carried out with a centrifugal force of 800 x g. Immediately thereafter, the supernatant was cold-bundled at -80 °C. Samples were thawed at 37 ° C and analyzed in a sensitive and specific commercial immunoassay within 1 hour. Standards and sample lines were subjected to two replicate measurements and the average was calculated. The result is multiplied by the dilution factor used for the sample.

a-PDGF-AB PDGF-AB was assayed using a Quantikine ELISA kit (#.DHD00B, R&D SYSTEMS, Minneapolis, MN). The sample was diluted 100-fold with ❹ Calibrator diluent (RD6-11). The wells were incubated for 2 hours, washed, and conjugated with the enzyme conjugated PDGF-AB antibody for further 3 hours at room temperature. Use Wash Buffer - to clean the wells, then add the substrate at room temperature (Substrate

Solution) 20_30 minutes. The hole is protected from light. Stop solution was added to each well and the absorbance at 450 nm was measured using a microtiter plate reader. The minimum detectable dose was 1.7 pg/ml. b-TGF-fil

TGF-βΙ was determined using a Quantikine ELISA kit (DB100B, R&D 34 200930726 SYSTEMS). The sample was diluted 1 〇〇 with calibrator diluent (RD5-26). A dilution sequence of TGF-βΙ standard (890207) having a volume of 1〇〇_μ1 was prepared in a microtiter plate coated with a TGF_p receptor π. Acid activation and neutralization reactions were performed prior to TGF_pi analysis to activate latent TGF_pi to an immunologically active state. For this purpose 'mix 0.5 ml of sample with 1.1 ml of in HC1 'mixed at room temperature for 10 minutes' Add 0.1 ml of 1.2N NaOH / 0.5M HEPES (N-[2-hydroxyethyl] 〇辰Morphine _n〇_[2-ethanesulfonic acid]) ❹ (Slgma 'H-7523) was neutralized and centrifuged. The total TGF-β 1 content of the supernatant fraction was then determined. Aliquots (5 μ μΐ) were added to the microtiter plate in duplicate, then capped and incubated for 2 hours at room temperature. After that, the wells were washed, and TGF-pi polyclonal antibody, which was lightly bound to the enzyme, was added, and cultured for 5 hours at room temperature. As described above, the detection limit of TGF-βΙ is 4 61 pg/m

c-EGF EGF was determined using a Quantikine ELISA kit (#.DEG00, R&D ❹ SYSTEMs, Minneapolis, MN). The sample was diluted 2 times with Calibrator diluent (rD6n). Add 20 μl of the standard * reference, reference or sample to the well. The well plates were incubated for 2 - hour at room temperature. Each well was suctioned and filled with a wash buffer for cleaning. EGF conjugates were added to each well and incubated for 2 hours at room temperature. The wells were washed with a washing buffer, and a solution (200 μM) was added to each well. The mixture was incubated at room temperature for 2 minutes in the dark. A stop solution (50 μM) was added to each well. The optical density of each well is a VersaMaxTM microplate reader (VersaMaxTM microplate reader,

Molecular Devices, USA ) Tested at 450 nm in 30 minutes 35 200930726. The minimum detectable dose is 0.7 pg/ml. d-IGF-1 IGF-1 was quantified using a Quantikine ELISA kit (DG100 from r&d SYSTEMS). The sample was diluted 100-fold with Calibrator diluent (RD5-22). As stated by the manufacturer, the minimum bond dose ranged from 0.007 to 0.056 ng/ml with an average MDD of 0.026 ng/ml. 15 μ μl of the assay diluent (RD1-53) was added to each well, followed by a 50 μΐ standard (890775). The well plate was covered with a strip of glue and incubated at 2-8 ° C for 2 hours. The wells were washed 3 times, and then IGF-1 combined with the enzyme was co-cultured at 2-8 ° C for 1 hour. The measurement was completed as described above. 1.7-Statistical Analysis The data for all series of experiments is presented as mean, standard deviation, and minimum and maximum values. A statistical comparison was made with a tw〇_tailed paired Student test. A p-value less than 0.05 is used to estimate the significant difference in mean PGF concentrations in the different steps of the plate preparation process (the significance 〇f heart)

Differences). The value is expressed as insignificant (Ns, < 〇 〇 5), < 〇 〇 1 or < 0.001. When the value is close to 〇·〇5, the exact p value is displayed. The preparation and separation of samples comparing I.8_PC, S/D-PC and Act_PC correspond to the initial platelet thick solution (pc), treated with solvent/cleaner (1% ΤηΒΡ and 1% Trit〇n χ_45). Samples of the initial platelet thick solution (S/D-PC) and the CaCl2-activated starting platelet concentrate (Act-PC) were separated by a dish to compare their 36 200930726 protein content. 20 pg of each sample was mixed with 5 pl NuPAGELDS sample buffer (4x) (Invitrogen), 2 μΐ NuPAGE reducing agent (10x) (Invitrogen) and deionized water to give a final volume of 2 μμ1 (activated jk platelet) The sample for the thick solution is 21 μΐ). The resulting mixture was then heated at 70 ° C for 10 minutes and subjected to SDS- • PAGE using a 4-12% polyacrylamide gradient gel (NuPAGE Bis-Tris, Invitrogen). f) Protein separation was performed at a constant voltage of 200 V for 35 minutes with a predetermined current of 150 mA/gel. The resulting gel was stained with Coomassie blue R-250. The protein-labeled Mark 12 unstained standard (Invhr〇gen) is used to measure the molecular weight of the protein in the sample. The Mark 12 label was loaded onto a NuPAGE novex 4-12% Bis-Tris gel (Invitrogen) with MES and separated by Coomassie Blue R 25 毕. The corresponding results are disclosed in the fourth figure. μ Eggs from the initial platelet thick solution treated by the solvent/detergent method. Self-map (Ρπ) ί!1〇 shows that it is not significantly different from the untreated initial platelet concentrate, but activated. The protein profiles of the plate and the thick liquid are obviously different. The bands in the 40 to 70 kDa region disappear. These bands correspond to the α, p and subunits of fibrinogen, which are respectively 63.5. , 56 and 47 kDa. 1.9 - Growth factor activity measurement In order to determine whether the growth factor obtained by platelet S/D treatment (1% ΤηΒΐΜ% τ_η χ _45) can maintain activity, a human osteoblast-like MG-63 cell line was used for in vitro cell culture studies. 37 200930726 MG-63 cells were treated with growth factor concentrates obtained from S/D-PC or from Act-PC, and cell responses were assessed by cell morphology and survival. 105 to 106 cells were cultured in a 35-mm Petri dish using 90% Minimum essential medium Eagle (MEM) with 2 mM-face acid, Eagle's BSS* ( Balanced Salt Solution), Adjust to 1.5 g/L sodium bicarbonate, 0. 1 mM non-essential amino acid, 1.0 mM sodium pyruvate, 10% 0 heat deactivated fetal bovine serum, and optionally with 5% S/D-PC or

Act-PC Growth Factor Thick Liquid. After culturing in a humidified atmosphere containing 5% CO 2 and 95% air at 37 ° C, the cells were washed with phosphate buffered saline (PBS, Gibco, UK), followed by trypsin-EDTA solution (0.25% trypsin) at 37 The cells were allowed to detach for 5 minutes at °C, then centrifuged and suspended for further cell testing. The live/dead cells (survival assay) were detected using the 3-(4,5-dimercaptothiazol-2-yl)-2,5-diphenyltetrazole rust bromide (MTT) assay. Electron microscopy was also performed to study cell morphology. Ο 3-(4,5-Dimercaptothiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazole rust (MTS) Assay to monitor cell-proliferation. Briefly, 500 μM of MTS (Celliter - 96 PromegaCorp USA) was added to each sample. After 60 minutes of incubation at 370 C, the absorbance at 490 nm was measured using a microplate reader. 1.10-Virus Deactivation assay The study of the efficacy of the solvent and/or detergent treatment for the initiation of platelet thick liquor lysis is carried out on a lower scale experiment based on international benchmarks such as EMEA and WHO recommendations. 38 200930726 ΐ Immune 1 small plate thick liquid 搀 human-associated virus (ΗΙν, human toxic abdomen floating liquid: bovine rabies Γ ― - poisonous model; fake, sometimes two T ^ V) ' in the absence of other convenient in vitro model virus leaves % was used as a model for HBV; a mixture of 1% ΤηΒΡ and 1% _ was stored at a high titer of the virus. The kinetics of activation were measured by different in vitro cell cultures during the time of Ο 及 and during treatment. Perform ^ _ to / then 疋 sense wood force, the data is TCID5 〇 / ml = residual virus infectivity. The resulting =: 7:: : = trace is > 5.6 ~ phase one main logl 〇 ' * in vsv for > ;7 〇'〇. So solvent and / or treatment will ensure It may be present in the lipid envelope virus of the initial platelet thick cell product towel when the + remaining virus is deactivated. L11_ expired frozen platelet growth factor composition = expired (more than 5 days after collection) platelet thick liquid is transferred to -20. Ϊ ί store for one month. Then they are unbundled in a 35 ° C water bath. The same experiment as fresh platelets is performed. The growth factor content of multiple preparations is measured. The corresponding results are shown in the fifth and the first Figure 6 1.12 - Blood clot formation assay Growth factor 39 derived from S/D treated initial platelet concentrate 2009 30726 Thick liquid system # is extracted by oil to remove _ and clear _ after recovery. 5 ml The resulting growth factor was introduced into a 5 n^ cartridge by thick liquid. 5 μM of 500 IU/ml bovine thrombin was introduced into another 5 mi syringe. The two syringes were placed in a double syringe connected by a single nozzle. Supply the device. Let the two components pass through. The plate gel will be in 5 seconds ^ II. Results

❹ ΙΙ·1-cell count = ten platelet separation studies obtained from ten different donors. The platelet thick solution obtained from platelet separation = '064 ± 235.2 X (10) platelets / ml (range: 782_ 1358 X 1 platelets / ml), the average WBC count is 〇ιΐ25 + 0.025 X Μ / ml ( Range: 〇〇7), — (10) ν, ±0·025 χ 1〇6/ml. Set II.2-Growth factor content storage lD=IGF_P1, coffee and coffee 1 in a variety of to, plate two preparation:: average concentration soil standard deviation (SD), minimum and maximum, to the two flute: Table 1. Individual data points from each series of experiments can be found in the second figure. 200930726

PDGF-AB (N=10) TGF-βΙ (N=10) EGF (N = 8) IGF (N = 6) m 13.8 16.6 <0.0007 83.4 A SD 14.3 14.3 - 32.8 A Min. 2.2 1.1 60.1 Max. 49.5 36.0 - 162.7 m 184.4 192.2 2.2 88.4 r> SD 80.2 37.4 1.6 33.5 J3 Min. 107.7 140.4 0.7 68.8 Max. 392.6 272.6 5.0 170.1 m 84.6 63.8 0.9 117.2 SD 35.5 14.1 0.6 34.9 U Min. 52.8 48.6 0.2 82.3 Max. 209.5 88.5 1.6 195.3 m 88.3 68.6 1.40 112.4 ΤΛ SD 45.9 27.2 1.0 39.7 xJ Min. 52.0 38.3 0.5 73.0 Max. 209.5 132.1 3.0 202.1 B vs A < 0.001 < 0.001 < 0.05 0.025 C vs A < 0.001 < 0.001 < 0.05 <0.001 P value BvsC < 0.001 < 0.001 0.044 < 0.001 B vs D < 0.001 < 0.001 0.087 < 0.01 C vsD NS NS NS NS 〇 Table 1: In the starting platelets (A), at l %TnBP-l%TritonX-45 After treatment (B), and after CaCl2 activation (C), the release agent was subjected to 1% TnBP-1% Triton X-45 treatment (D) for the average concentration of growth factors, (ng /ml). NS: No significant difference. The average PDGF-AB content in the starting platelet concentrate was 13.8 ± 14.3 ng/ml (N=10). After direct S/D treatment with TnBP-Triton X-45, its content increased significantly (p<〇.〇〇1) to 184.4 ± 80.2 ng/nu', which is approximately 13 times greater than the initial platelet concentrate. When the initial platelet concentrate is first activated by CaCl2, its content 200930726 will also increase significantly (84·6 ± 35·5, p < 0.001), but the increase is less (about 6 times), and it is in the subsequent s The /D process remains substantially unchanged (88.3 ± 45.9, NS). In the unactivated S/D treated platelets, the PGDF-AB content was significantly higher than that in the activated, and activated/S/D treated platelets (p<〇.〇〇1). There are similar data on TGF-βΙ. The average TGF-βΙ content in the starting platelet thick • thick solution was 16.6 ± 14.3 ng/ml

(N=10). After direct s/D treatment, the TGF-βΙ content increased by nearly 12-fold than the initial PC ’ to 192.2 ± 37.4 ng/ml (p<0.001). When the starting platelet concentrate is first activated by CaCl2, the content is increased by about 4 times (63.8 ± 14.1 ng/ml) (p<〇.〇〇l) and remains in a distinctly different state during subsequent S/D treatment. (68.6 ± 27.2 ng/ml). Further, in the S/D-treated platelets, the average content of TGF-βΙ was significantly higher than that in the activated and activated S/D-treated platelets (p<0.001). EGF (<〇7 pg/ml) could not be detected in the initial platelet thick solution, but became detectable after S/D treatment, and the average EGF ❹ content (2.2 ± N = 6) was significant. Higher than (p<0.05) the value obtained after CaCl2 activation (〇·9 ± 0.6 ng/ml) (p<〇.〇5), or apparently after the activation of CaCl2 followed by s/D treatment ( 1.4 soil ~ 1.0 ng / ml). In the starting platelet concentrate, the average IGF-1 content was 83.4 soil 32.8 ng/ml (N-8). Compared to other platelet-derived growth factors, it did not appear to have a significant increase after S/D treatment (88 4 ± % 5, p = 0.025). In platelet preparation after CaCU activation, its average content was even slightly higher (P<〇.001) (117.2 ± 34.9 ng/ml) and remained stable after subsequent S/D treatment (112.4 ± 39.7 ng/ml) ). 42 200930726 In 150 ml of platelet concentrate, the total amount of PDGF-AB, TGF-βΙ, EGF and IGF-1 recovered after direct S/D treatment (153 ml) were 28213, 29406, 336 and 13525 ng, respectively; After CaCld^, they were 10152, 7156, 108, and 14064 ng, respectively; the 20% average volume loss caused by platelet gel formation (120 ml) was also counted. This confirms that the S/D treatment is more effective in releasing PDGF-AB, TGF-βΙ and ^ EGF than CaCl2 activation. Table 2 compares the platelet-derived growth factor in the initial fresh plate of concentrated φ thick solution (A), after centrifugation (centrifugation into a mass and removal of platelets) followed by TnBP-Triton X-45 treatment of the supernatant Time (B), and after S/D treatment of the initial platelet thick solution with 1% ΤηΒΡ-1 % Triton X-45 (C) or after S/D treatment with 2% TnBP (D) . The levels of PDGF-AB, TGF-βΙ, EGF and IGF-1 in untreated platelet concentrate (Α) were consistent with previous data (Table 1). When the platelet-free supernatant (Β) was treated with TnBP-Triton®-45 (Β), the content of PDGF-AB, TGF-βΙ or EGF remained low or could not be detected (3.9; 1.1; ρ<〇. 〇〇1) 'The content is high, but similar to the amount in the sputum starting platelet concentrate (75.4 vs. 72.2 ng/ml). Therefore, PDGF-AB, TGF- (31 and EGF release during S/D treatment - depends on the presence of platelets. Platelet solution treated with 1% TnBP-l% TritonX-45 or 2%, ΤηΒΡ In the cell product, the increase in the content of platelet-derived growth factor is similar. Further, after the platelet concentrate is treated with 1〇/〇tritonX-45 or 1%trit〇nX-100, the subsequent detergent treatment will be in the absence of oil. In the case of removal by tC 18 adsorption (sp 1 T45tC 18 and SPlT100tC18); this shows that the growth factor PDGF-AB observed after treatment with 2% ΤηΒΡ or 1% ΤιιΒΡ and 1% TritonX-45 The release of TGF-βΙ, IGF and EGF is within a range of 43 200930726. Therefore, the extent of platelet lysis is essentially the same as the use of this solvent and detergent combination or solvent or detergent alone. The final 'data (not shown) indicates that 'centrifugation of the sample at 10000 X g or 800 X g does not affect the amount of platelet-derived growth factor measured in this assay. PDGF-AB TGF-pl EGF IGF-1 A 2.9 1.1 <0.0007 72.2 B 3.9 1.1 <0.0007 75.4 C 128.7 207 .9 0.8 79.9 D 133.7 185.6 1.2 89.8 Table 2: Multiple growth factors in the starting platelet concentrate (a), centrifuged platelet-free supernatant after TnBP-Triton X-45 treatment (B), at TnBP- Concentration (ng/ml) in the initial platelet concentrate after Triton X-45 treatment (C) or after 2% ΤηΒΡ treatment (D). Further, fibrinogen, fibronectin, albumin, The concentration of immunoglobulin IgG and II, VII, VIII, IX, X, XI, XIII coagulation factors and Van derrick's factor are in S/D-treated platelet concentrate and CaCl2-activated platelets. The thick liquid was measured in the growth factor thick solution. These results are shown in Table 3. 〇44 200930726 Protein S/D-PC Act-PC Fibrinogen (g/L) 2.6 0.2 Fibronectin (g/L) 0.33 0.12 Factor VIII (IU/ml) 0.92 <0.1 Factor II (IU/ml) 1.2 <0.1 Factor IX (IU/ml) 1.02 <0.1 Factor X (IU/ml) 1.15 <0.1 Factor VII (IU/ml) 1.08 <0.1 Factor XIII (IU/ml) 0.87 <0.1 Factor XI (IU/ml) 1.35 <0.1 Van Wyber's Factor (IU/ml) 0.95 <0.1 albumin (g/L) 33.8 32.5 IgG (g/L) 11.5 11.2 Table 3: Starting platelet concentrate obtained from 1% TnBP-1% Triton X-45 treated with the present invention (S) /D-PC), or a comparison of various protein concentrations in a growth factor concentrate obtained from CaCl2-activated starting platelet concentrate (Act-PC). Therefore, it is shown that the activated platelet concentrate almost completely removes fibrinogen and clotting factors. II.3-Bovine thrombin activation experiment

CaC^/glass pellet treatment only partially activates platelets, which may explain the lower levels of PDGF-AB, TGF-pl and EGF in the release than in the S/D lysate; in order to break the above assumptions, Therefore, the content of these platelet-derived growth factors in the release of platelet thick liquor 45 200930726 (third image) was determined, wherein the release of the aforementioned j6l platelet thick solution was first activated by CaCl2, and then received bovine thrombin (Act- Further treatment of T) and thrombin and further S/D treatment (Act-TS/D). The third panel shows: PDGFAB (A), TGF-βΙ (B) and EGF (C) in the initial platelet concentrate (start), after CaCl2 activation (Act) followed by bovine thrombin activation (Act- T) and S/D treatment (Act-T-SD) • The content of the time. It can be seen that, in contrast to CaCl2 activation, the addition of bovine thrombin does not increase the release of PDGF-AB (A), TGF-pi (B) and 10 EGF (C). This is the first time that 'release of platelet growth factor (especially the release of PDGF-AB, TGF-pl and EGF) when platelets are S/D treated is more effective than calcium and/or thrombin to activate platelets. It is much higher. S/D treatment should induce the dissolution of lipid-rich platelet membranes, and release platelet-derived growth factors from intracellular alpha particles. The fact is that when thrombin activation is first performed, the release of platelet-derived growth factors may be lower, probably because agglutinated platelets and partially released platelet-derived growth factors are caught in the fibrin network (called In platelet sputum, the network is formed by the polymerization of fibrinogen in the starting platelet concentrate by thrombin. Indeed, CaQ2 • only causes partial activation, and the formation of blood clots caused by endogenous thrombin. * Unexpected, menstrual, can be further added to the activated platelet release to about 48 NIH. After bovine thrombin/mi, it still does not cause the fact that the release of platelet-derived growth factor continues to increase. Complete activation of platelets can also be confirmed by the extremely low (or undetectable) concentration of fibrinogen and clotting factors, as shown in Table 3 above. Similarly (not shown), we found that the platelet growth factor content in the effluent was similar when using bovine thrombin activity 46 200930726 platelet concentrate. We also found that the IGIM content was retained after s/D treatment, and the quality was unchanged, but only slightly increased after the activation of the part/thrombin. This, 'σ fruit can also reflect the vast majority. It is already in the form of free circulation in the mortar - the fact that only a very small proportion is present in the platelets. Our data show that, compared to other methods such as thrombin activation, cryo-thaw cycles and/or freeze-drying, S/D treatment is used to effect platelet lysis by platelet growth factor from platelet cells. The most effective means of releasing the particles into the exudate. Earlier studies have reported an average amount of PDGF of 17 5 ng/ml in whole blood serum, which is 0.06 ng per 106 platelets. This amount is equivalent to 6 ng/m b of the separation platelet concentrate in our study and we found that the amount of pdgf in the S/D treated gold plate thick solution was on average about three times higher. Our data further show that to increase the release of pDGF_AB, TGF-βΙ and EGF from platelets, 1% TnBP_1% can be used.

Triton X-45 combination, 2% ΤηΒΡ, or 1% triton x_45 or Triton 〇X·*100 S/D treatment, and oil extraction to remove S/D, does not reduce gold plate dissolution Comparison of the content of four kinds of platelet growth factors in the cell product. II. The lipid content in the growth factor thick solution. The lipid in the platelet growth factor thick solution after the initial platelet thick solution was activated by S/D treatment or CaCl2 activation. The content results were measured and compared to the levels in the starting platelet concentrate, as shown in Table 4. 47 200930726 Triglyceride (mg/dl) HDL (mg/dl) LDL (mg/dl) — 170 33 99 162 ----- 101 62 20 ----— 11 0 43 ---—______ 2 0 156 ----------- 31 97 Cholesterol Startup PC Trit-PC S/D-PC 1 S/D-PC 3 164 —--- 160 150 Ο Lipid content liquid preparation growth factor Thick liquid oil extraction (10) H Department secret' followed by three times of oil extraction (s = this ^ 3 J after the 'after proceeding. C3), or CaCb activation in platelets. Each subject was tested by HltadU clinical technical instrument. The system has a pH of 7 2 and contains 88" 1 〇 3 blood ❹ small plates μ, Ο · 1 X 1 〇 3 white blood cells (WBC) / Μΐ and 〇·〇8 χ 1〇6 red blood cells (RBC) / Ml of the same starting also a small plate of thick liquid to prepare. ' As shown in Table 4, compared with Triton-treated platelets or activated platelets LDL (low-lipoprotein), HDL (high-density lipoprotein), triglyceride, and linalool in the S/D-PC growth factor concentrate prepared by S/D treatment. The amount is significantly lower. Further, no significant difference is observed in the extraction of the lysed blood plate by S/D with oil extraction for one or three times. / Cholesterol, triglyceride in the growth factor thick solution of the present invention The removal of vinegar and LDL will lead to a major cause of clinical and therapeutic goals, 48 200930726 because triglycerides and LDL-cholesterol combinations are known to be atherosclerotic and form fatty plaques in the arteries and venous systems. The role played by the development of cardiovascular disease, these problems Causes heart attack, stroke and peripheral vascular disease. Nippon.5-Growth factor activity microscopic observation shows the shape, size and number of cells: when the cells are concentrated with S/D or CaCl2 activated platelets A large number of spindle-shaped cells are particularly observed when co-cultured with the blood-small (4) growth® sub-concentration. Further, the cell count is not 'co-cultured with Act-PC or with growth factor concentrate. Compared with cells, when the cells were co-cultured with SD_PC growth factor thick solution, the number of MG-63 osteoblasts increased significantly. The results of MTT analysis also showed that compared with Act p (: treated cells) The presence of SD_PC growth factor concentrate increases the cell viability of cultured cells. S/D_pc or Act_pc growth factor concentrate does not exhibit cytotoxicity to MG-63 osteoblasts. 故 Therefore, the results of this study show that S/D treated platelets or

The growth factors obtained from both activated platelets remain active after the selected extraction method. However, our experiments show that when cells are incubated with S/D, a growth factor-rich thick solution, the cellular response increases significantly. The method of the present invention (4) has an improved effect on the cells of the (6) cells, and may now be due to an increase in the growth factor content, particularly PDGF, TGF_P1 and coffee (these factors are important in wound healing and tissue regeneration). The sexual system is known, and/or may be due to an increase in the amount of other biologically active substances that are not present or have a low amount of expression in the activated platelet concentrate. 49 200930726, these results also confirm the important potential of the present invention or the growth factor concentrates afforded by the method of the invention in terms of therapeutic use and in the preparation of modified cell culture media. II.6 - Virus Deactivation In addition, there is ample evidence that the S/D treatment used here effectively deactivates blood-borne envelope viruses, particularly Hiv, hbV and - HCV. 1 1% TnBIM% Triton X-45 combination as shown in the materials and methods above

Treatment at 31 °C will ensure that the HIV, BVDV and PRV reduction factors are >5.6, >6.6, and >6.4 log10 within 5 minutes after treatment, and will also quickly 27.0 l〇gl() The VSV and Sindbis model virus are deactivated. Non-enveloped viruses (such as HAV and small virus B19) cannot be deactivated by S/D treatment, but they are only pathogenic to a small number of patients with altered immune function. No pooling can significantly reduce the statistical risk of contamination of a single donor's allogeneic platelet-derived growth factor formulation, but further nanofiltration can also be used to reduce the risk of non-enveloped virus contamination. Obviously, the platelet-derived growth factor preparations described herein will have to be produced under Good Manufacturing Practices (GMP) conditions, such as guidelines for viral inactivation and removal procedures intended to assure the viral safety of human blood. Plasma products. www.WHO.int. Geneva, 2003: 1-72). The S/D treatment of such jk platelets provides a degree of practical advantage in topical applications. First, the safety of viruses transmitted by allogeneic organisms will improve, giving these products a broader clinical potential. Second, the release of higher potency platelet-derived growth factors by S/D treatment may improve the procedural cost-effectiveness of using 2009. Third, in the case where recombinant platelet-derived growth factor is not allowed, the virus deactivation method can make the allogeneic platelet-derived growth factor more convenient, or, in the case of allowing the use of recombinant platelet-derived growth factor (such as To treat certain lower extremity diabetic ulcers, when used in combination with natural platelet-derived growth factors, a beneficial synergistic effect can be obtained. Finally, the virus-deactivated platelet-derived growth factor can be incorporated into a fibrin-based artificial eagle as a topical application to accelerate healing, as a substitute for fetal cattle, or in cell engineering studies. Or a mesenchymal stem cell in vivo expansion (exjpansi〇n) and its differentiation into osteoblasts or chondrocytes as a recombinant platelet-derived growth factor. [Simple description of the diagram] The first circle: study design: the isolated platelets (N=8) were divided into two subset pools. Subset pool 1 was treated directly with 1% TnBP-1% Triton X-45 for 1 hour without first being activated with CaCl2. Sub-pool 2 was first activated with 23 niM CaCL and glass pellets to form a platelet gel. After 1 hour of incubation, the blood clots were removed and the liberated material was treated with 1% TnBP-1% Triton X-45 for 1 hour. The solution treated with both S/D (solvent-detergent) was subjected to oil extraction (3 times) to remove the solvent and detergent. The growth factor content of the starting platelets, after activation, and after S/D treatment-oil extraction was measured. Second circumstance: (A) PDGF-AB, (B) TGF-βΙ, (C) EGF and (D) IGF in the initial platelet concentrate (start), in direct S/D treatment (1% TnBP- After 1% Triton X-45) (S/D), after initial platelet concentrate was activated by CaCl2 (activation), and after CaCl2 activation 51 200930726, platelet release was treated with S/D (1% ΤηΒΡ- 1% TritonX-45) Post (activation + S/D) content (ng/mi). The s/D treatment was carried out as described in Materials and Methods and included 3 oil extractions to remove the S/D agent. The third circle: (A) PDGF-AB, (B) TGF-βΙ and (C) EGF in the initial platelet thick solution (start), after the initial platelet thick solution is activated by CaCl2 (Act) followed by cattle Content (ng/ml) when thrombin activation (Act-T) and 'S/D treatment (1 % ΤηΒΡ-1 % Triton X- 45 ) (ACT TS/D ). The S/D treatment is performed as described in Materials and Methods and includes 3 oil extractions to remove the S/D agent. Fourth circle: between the starting platelet thick solution (line 1), the same platelet thick solution treated by the solvent-cleaner method of the present invention (line 2), and the same platelet thick solution (line 3) after activation of CaCls (line 3) Comparison of protein compositions. Samples were separated by SDS-PAGE on 4%-12% gels, after which the proteins were stained with Coomassie blue. The μ line corresponds to the protein label (size in kilodaltons, indicated on the left side of the gel). Figure 5: Excessive cold bed platelets are treated with S/D (1% ❹ TnBP_l% Triton X_45) (S/D-PC), or CaCl2 activation (Act-PC), or selective activation in CaCl2 followed by s The composition of PDGF-AB (figure a) and EGF (figure b) was compared in the thick solution obtained by /D treatment of k (Act-PC+S/D). Figure 6: Overdue frozen platelets are treated with S/D (1%)

TnBP-1% Triton X-45) (S/D-PC) or a concentrated solution obtained by CaCl2 activation (Act-PC), IGF-1 (sixth panel a) and TGF-βΙ (sixth panel b) Comparison of the composition. [Main component symbol description] Benefit 52

Claims (1)

200930726 X. Patent application: h-type clotting platelet growth factor thick solution for therapeutic and/or cosmetic use. 2. The condensable platelet growth factor thick liquid as described in claim 1 which comprises the growth factors PDGF, TGF-β, IGF, EGF, CTGF, bFGF and VEGF. 3. Condensable platelet growth factor concentration concentrate as described in claim 1 or 2, which is characterized by the absence of blood cell-related blood transfusion reaction. 4. The condensable platelet growth factor thick solution according to any one of claims 1 to 3, further comprising at least one selected from the group consisting of fibronectin, vitronectin, A protein consisting of a thrombin-sensitive protein (thr〇mb〇sp〇ndin), a von Willebrand factor, and a group consisting of II, V, VII, VIII, IX, X, and χι coagulation factors. 5. A method for preparing a clotting platelet growth factor thick solution, the method comprising the steps of: • a) contacting the starting platelet concentrate with a solvent and/or a cleaning agent; • b) causing the aforementioned initial platelet thick solution And co-cultivating the solvent and/or detergent together at a pH ranging from about 6.0 to about 9. 及 and at a temperature ranging from 2 ° C to 50 ° C for at least 5 minutes to 6 hours; And c) removing the aforementioned solvent and/or cleaning agent by oil extraction and/or chromatography. The method of claim 5, wherein the solvent is selected from the group consisting of di- or trialkyl phosphates, di- or di-alkyl-disc acids having different alkyl chains. Group of groups. 7. The method according to claim 6, wherein the solvent is tri-n-butyl phosphate (ΤηΒΡ). The method according to any one of claims 5 to 7, wherein the cleaning agent is selected from the group consisting of a polyoxyethylene derivative of a fatty acid and a partial esters of sorbitol. A group consisting of nonionic detergents, sodium decodeoxycholate and sulfobetaines. 9. The method of claim 8, wherein the cleaning agent is Triton X-45, Triton X-100 or Tween 80. The method according to any one of claims 5 to 9, wherein the respective final concentration of the solvent and/or the aforementioned cleaning agent ranges from 0.2 to 5% by volume, which is thickened by the aforementioned initial platelets. The volume of the liquid is calculated based on the basis. 11. The method of claim 5, wherein the platelet © thick solution is contacted with 2% ΤηΒΡ alone or with 1% ΤηΒΡ and 1% Triton Χ-45, which is initiated by the foregoing The volume of the platelet thick liquid is calculated on a volume basis. The method of any one of claims 5 to 11, wherein the oil extraction is carried out with a pharmaceutical grade oil, the amount of the oil being from = to 20% by weight, or from 5 to 15 parts by weight. %, or from 5 to 1 〇 篁 / 〇, based on the weight of the mixture of the platelet thick solution and the aforementioned solvent and / or detergent. The method of any one of clauses 5 to 12, wherein the chromatographic method comprises a C18 bismuth telluride filling material, or 54 200930726 14. 15. ❹ 16· 17. 18. Ο 19. SDR (Solvent-Detergent Removal) The method described in any one of claims 5 to 13 which further comprises step d), using a 10 to 75 The nanoporous membrane was subjected to nanofiltration. The method of any one of claims 5 to 14, further comprising a preliminary step of preparing a starting platelet thick liquid prior to the aforementioned step a). The method of claim 15, wherein the initial platelet rich solution is prepared from whole blood by apheresis or buffy-coat separation. The method of any one of claims 5 to 16, wherein the aforementioned initial platelet rich liquid is in a fresh, expired, or expired and frozen state. The method of any one of claims 5 to 17, which is used for the therapeutic and/or cosmetic use according to any one of claims 1 to 4 of the patent application. Condensable platelet growth factor thick solution. A method of forming a blood clot, which is a condensable platelet growth factor thick solution obtained by the method according to any one of claims 5 to π, or as claimed in claim 1 The clotting platelet growth factor concentrate for therapeutic and/or cosmetic use according to any one of the four items is mixed with thrombin. The method of claim 19, wherein the activity ranges from 20 IU/ml to 1 〇〇〇iu/ml and 1 volume of the aforementioned clotable platelet growth factor Thick liquid mixing 〇 55 20. 200930726 ❹ ❿ 21. The method of claim 20, wherein the aforementioned coagulation is human thrombin. a medicinal product comprising a condensable platelet growth factor thick solution obtained by the method of any one of claims 5 to 10, or as claimed in claims 1-4 to 4 A clotting platelet growth factor concentrate for therapeutic and/or cosmetic use as described. A clotting platelet growth factor thick solution obtained by the method of any one of the items 5 to 17 of the patent application, which is used for therapeutic treatment according to any one of claims 1-4 to 4 And/or the use of coagulated platelet growth factor for cosmetic use, which is used to form blood clots. A condensable thick liquid of platelet-derived growth factor obtained by any one of the items 5 to 17 of the patent application, ^ The clotting platelet growth g] used for the treatment of sputum and/or smear, as described in any one of the four items, for the regeneration of the bone path, or for the (four), woven and hard tissues. - for use in a physiotherapy and/or beauty treatment as described in any of claims 1 to 4, as claimed in any of claims 5 to 17 of the patent application. Condensable platelet growth factor for sexual use; = , use 'f for cell culture in vitro studies Ov"ro) or in vivo studies 〇χν/ν〇). 22. 23. 24 25. 56