TWI344963B - Clottable concentrate of platelet growth factors and preparation method thereof - Google Patents

Description

1344963 IX. Description 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 thick fluids 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 tissue 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, 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 by the formation of a blood clot (cl〇t), which is characterized by fibrin and multiple proteins that crosslink each other. Such as vitronectin, fibrectin, and thrombospondin, and this blood clot prevents re-bleeding and acts as a substrate for invading cells while providing a resistance The barrier to pathogen invasion. In addition, this initial gold 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 trans-receptors (reCept〇r) functional domains (d〇main) across the cell membrane. Real physical interactions to adjust cell function. Later, the secondary message transducer controls the multicellular biology of cell decay 5 1344963. Although only a portion of the roles and mechanisms involved in tissue regeneration are currently being interpreted', most of the potential benefits have been demonstrated, particularly in the advantages of platelet derivatives, especially platelet-derived growth factors ( Platelet-derived growth factor) advantages. 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, extracellular) There is a direct correlation between matrix deposition and remodeling) and enhanced cell proliferation. Further, platelet derivatives are also indirectly related to some closely related biological functions, such as chemokine by bystander cells such as fibroblasts, macrophages, epithelial cells or lymphocytes. Production of cytokines. Therefore, platelet-rich preparations, as well as platelet gels, viscose and their eliminators, are used alone or in combination with transplanted biomaterials.

Regeneration and wound healing gradually gradually pay attention to and support the avoidance of UxWvo) amplification and differentiation of endothelial cells and fibroblast proliferation 3 temples, so its therapeutic advantages in cartilage σ are gradually affected by 6 1344963 local blood • Preparation of small plate source preparations (such as gels, gels, and eliminators) - usually by mixing platelet concentrate with an activator that triggers the release of platelet granules to mimic the physiological activation of platelets. The activation step is generally carried out by the direct addition of exogenous thrombin or by the use of calcium chloride (CaCls), which counteracts the effect of adding anticoagulant during blood or blood-small plate collection, which triggers blood agglutination. A coagulation cascade and release of endogenous thrombin. Endogenous. Sexual and exogenous thrombin also induces the polymerization of fibrinogen and forms fibrin-based biomaterials (blood clots), resulting in multiple combinations of platelet inclusions. 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) (Jassen Cilag Internat.) from a conventional expression system. 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, since I was able to observe the synergistic effect of the combination of multiple growth factors, the platelet source preparation still provided a supplemental advantage of φ compared to the use of a single recombinant factor. The main drawbacks of existing formulations containing platelets or platelet sources are that one of these formulations lacks proper standardization and definition, which can result in variations in the characteristics of platelet-rich products with multiple therapeutic effects. The simple hypothesis of using platelet concentrations to estimate the amount of growth factors in platelet derivatives is indeed unclear, as the techniques used to collect and/or activate platelets affect the growth factor content and the resulting clinical effects. The heterogeneity of the product may depend on differences in the following parameters: platelet concentration, white blood cells in the starting material, type of starting material, storage time, storage conditions, and manner of activation. Therefore, these variables apparently cause significant differences between products 7 ^ 44963 after the occurrence of the biological properties and clinical validity (such as healing power). For example, when a solution/glue containing intact platelets is directly delivered to the wound, the activation of the platelets is rapidly formed with the fibrin clot, and, similarly, with the incomplete dissolution of the platelets. Therefore, the complete platelets of ^ 1 are still encapsulated inside the fibrin clots' so it is difficult to determine the true release of growth factors. In the second step, when the platelet gel and/or growth factor preparation is activated by thrombin activation and platelet stimulation, when the platelets are known, the fibrinogen is completely depleted, no longer The clotting state 'Thus, it needs to be mixed with exogenous natural or synthetic products before being sent to the wound site.

In the "step", most of the therapeutic topical platelet derivatives are prepared from the source of the body. That is, the autologous platelet thick solution is prepared from the blood donated by the person in the hours or days before surgery. And use = focus care (p〇int_0f_care). Therefore, they are mostly used for planned surgery that requires a solid volume of platelet gel, as well as for patients who are healthy donors. In addition, the preparation of autologous preparations at the surgical site is There are disadvantages, because they are often carried out under good financial conditions, ^ lack of standardization to ensure growth factor release and its clinical validity. a, we have the method of allowing the preparation of platelet derivatives in advance Special 1 is the case of preparing platelet raw books and sub-concentrated liquids in advance under standardized conditions, so that a tailor-made agent can be prepared for various special physiological and pathological conditions. Step by step, existing platelets The other source of the source product is the important sea Sir Ran / there is an immune response of the intact blood cells or important blood cell debris to the donor antigen. Pin 4: X 8 1344963 may cause The serious consequences 'as observed in the gold-receiving reaction, and include the patient's autoimmune and hemolysis complications. Further, another major drawback of the existing platelet-derived products is the inevitable use of biological substances. Risk of related infections. Although in countries with a developmental management environment and blood collection services in the South, the safety of human allogeneic blood sap using a single donor to test a single donor is indeed high, despite standard blood bank methods (eg Apheresis or platelet preparation derived from whole blood. It is possible to prepare conditions for the second bacteria. However, when using a small plate derivative, virus and bacterial infection can occur. Therefore, platelet derivatives and The virus innocuousness of platelet growth factor is extremely important. The allogeneic platelet preparation used in the Ministry has the ideal virus. Finally, the increase in the elderly population and chronic diseases has become a serious problem. Although there are a variety of therapeutic treatments on the surface of the ^, including, for example, the hand 2 face:: Ϊ: Ϊ Ϊ 补 replantation, tissue wounds and groups intensive research The demand for ^, ^ Jizhi ingredients is still growing. The inventors have surprisingly discovered that these objectives can be achieved. Condensable platelet growth factor thick solution and its preparation are simple, rapid and effective for the preparation of platelets. Most people can obtain clotting blood from individual platelet thick liquid or from small plate. Cylinder 4 long factor thick liquid. More special 宗~^(4) Compared with Xiaobansheng, other methods in the field of this Λ日Λ方, at least significantly increase the growth of all growth factors θ 生==Recovery of important platelet growth factor' More special health (four) sub-PDGF, Qing-like EGF recovery. In the case of 9-step, the method of the present invention dissolves the lipid membrane, so the lipocapsid virus and other pathogens (such as fine 8 and parasites such as protozoa) are deactivated and the platelets in the starting platelet can be removed. Plasma and platelet lipids present in concentrated liquids. Therefore, the method of the present invention makes it possible to provide a virus deactivated Ί 凝, π Α Ι Ι, plate growth target concentrate, which can be effectively standardized for therapeutic treatment. Clinically, when platelet is used intravenously to treat quantitative or functional platelet count hypothyroidism (thr〇mb〇cyt〇penia), since the shelf life of platelets is 5 or 7 days (partly because of the risk of bacterial contamination), Therefore, a large number of platelet units with a storage time greater than 5 or 7 days are usually discarded each 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 concentrate for therapeutic and/or cosmetic use. In a preferred embodiment, the condensable platelet growth factor concentrate comprises growth factors PDGF, TGF-β, IGF, EC5F, CTGF, bFGF and VEGF. In another preferred embodiment, the aforementioned clotting platelet growth factor concentrate does not induce a transfusion reaction associated with jk fluid cells. 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 van derberg factor (v〇n).

Willebrand factor) is a group of proteins consisting of 凝血, V, VII, Vin, IX, X and XI 1344963 coagulation factors. Another object of the present invention is a method for preparing the present platelet growth factor thick solution, which can be used to contact the solvent and/or detergent in a thick plated liquid. 2 ° C to 50 ec Ming R... Main 乂υ I 巳 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少In the preferred embodiment, the above-mentioned solution or clear oil extraction and/or chromatography is used in the present invention, and has a group consisting of different St: classes; Qian Jia is a tri-n-butyl acid vinegar

In the method of the present invention, the detergents used in the method of the present invention 1pa: i fatty acid, polyoxyethylene derivatives, sorbitan partial S, beet ^ e / te =), _ sub Sex cleansers, deoxycholic acid steel and sulphide group (sulfGbetaines) group; and better group of 45 'TMtGn X_1GG and T_ 80 and / in ^ ^ 1 turn 3 The individual final concentration range of the aforementioned dissolution % detergent is from 〇·2 to 5 body thick liquid t Lin is from 02 to 2 volume 0/〇, which is based on the aforementioned starting platelet ί^ί ff Calculation. In the preferred embodiment of the method of the present invention, the starting platelet thick solution is used for 2% Tnm> blood transfusion and 1% THt〇n Χ·45 contact, which is the same as the blood plate; Volume based calculation. In the preferred embodiment of the present invention, the oil extraction is carried out in the form of medicinal /, /, and the foregoing oil is used in an amount of from 2 to 20% by weight, 11 1344963 or from 5 to 15% by weight, or from 5 to 1% by weight, based on the weight of the mixture of the aforementioned t-plate thick liquid and the aforementioned solvent and/or detergent. In another preferred embodiment, the chromatographic method is used, for example, comprising a C18 ceria filler material, or SDR (solvent_cleaner removal,

Solvent-Detergent removal) hyperD to remove the aforementioned solvents and/or cleaners. In a preferred embodiment, the method of the present invention further comprises the step of subjecting the resulting clotable platelet growth factor concentrate to nanofiltration using a 10 to 75 mm aperture filter or similar virus removal membrane. In a preferred embodiment, the method of the present invention comprises a preliminary step of preparing a platelet thick solution which is separated by platelet separation or buffy-coat. The method is prepared from whole blood and is in a state of being fresh or expired and stored in a liquid state, or expired and stored frozen. Another object of the present invention is a method for forming an A clot which is obtained by mixing the platelet growth and the thickening solution obtained by the method of the present invention with a thrombin. Preferably, the thrombin used in the aforementioned method of forming a blood clot is obtained from a human. In a particular embodiment, the amount of 0.1 to 1 volume of activity is from 2 〇 IU/ml to 1 〇〇〇 iu/ml of thrombin and 1 volume of the invention or obtained by the method of the invention. Condensed as a small plate growth factor thick liquid mixed. Another object of the invention is a pharmaceutical product or artificial scaffold comprising a condensable <RTIgt; platelet growth factor thick liquid of the invention or obtained by the method of the invention. U 12 Another object of the invention is the use of a cleavable platelet growth factor thick solution of the invention or by the method of the invention for forming a blood clot, or for bone regeneration or wound healing Or in vitro study (hv) or in vivo study (ex Wv0) cell culture \ [Embodiment] One of the objects of the present invention is a clotting platelet growth factor thick liquid, which is used for therapeutic and / or cosmetic use. The term "coagulation" as used in the present invention means that the platelet growth factor thick liquid obtained by the present invention or by the method of the present invention contains fibrinogen and XIII coagulation factor, so that there is a therapeutic application. When required, it can be mixed with a suitable activator (such as thrombin to form a blood clot. In a particular embodiment, the fibrinogen concentration in the clotting platelet growth factor concentrate of the present invention is preferably higher than丨 “Better than L5g/L thick liquid” is better than 2·5 2 = dry liquid, and its ΧΠΙ factor concentration is preferably higher than G.5 lU/ml, agriculture == high In the case of π51·dense liquid, it is more preferable that it is high (four) μ in the form of “beauty use” refers to the treatment of contact with various body parts, especially contact with the skin mucosa & 曱 嘴唇, lips, external reproduction 11 official, or with the teeth and mouth = touch, so that it is clean and aromatic, and has the effect of protection and change, or to maintain it in a good state. "Therapeutic use of therapeutic use or prevention in the present invention" Sexual treatment to deal with humans and snoring Recovering 'correction= 13 1344963 by medicinal, immunological or metabolic effects to change its physiological function. The coagulated 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 diseases, And/or for contact with the body part of the body, because it has been cleaned of both solvent and detergent, and is virally safe. y ' "Clearing" granules and / or detergent J means The solvent and/or cleaning dose in the condensable gold plate growth factor concentrate is extremely low, and preferably the amount cannot be detected. Indeed, it is known to those skilled in the art that the concentration of φ solvent and/or detergent is increased. High and long-term toxicity are directly linked, and in particular directly linked to the occurrence 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). The term "clearing solvent" in the present invention means that the amount of the solvent is less than 100 ppm', preferably less than 5 〇 ppm, more preferably less than 2 〇 ppm, still more preferably less than 10 ppm, less than 5 ppm, and More preferably, it is less than i ppm ° • Further, the term "clearing the cleaning agent" in the present invention means that the amount of the cleaning agent is less than 500 ppm, preferably less than 25 〇 ppm, more preferably less than 100 ppm, and even more preferably It is less than 5 〇 ppm, and even more preferably less than 10 ppm. "Virus safety" means that the "condensed platelet growth agent thick liquid is substantially free of infectious virus, especially without a lipid-containing virus", such as human immunodeficiency virus (HIV), for example. Hepatitis B virus (10) v), hepatitis C virus (HCV), western poison (WNV), TT virus, dengue virus, cytomegalovirus (CMV), Epstein Barr (EBV), human secret virus _8 (hhv 8 ), u 1344963 Colobus foam virus 'Severe Acute Respiratory Syndrome Virus (SARs Coronavirus) and other lipid envelope hepatitis viruses, cytomegalovirus, lactate dehydrogenase virus, Herpes group virus, baculovirus (rhabdovirus), leuk virus (leuk〇virus), mucus virus, alphavirus, arbovirus, paramucus virus, arenavirus, and coronavirus. "Substantially without" means that the degree of virus deactivation of the condensable blood and small plate growth factor thick solution is at least greater than 4 log10'. This is the standard set by the attenuating step of the strong virus (by the European Pharmaceutical Products Assessment Agency) (Committee for proprietary medicinal products (CPMP) (European Agency for the Evaluation of Medicinal Products, EMEA) is defined in the Note f〇r guidance on virus validation studies, references • CPMP /BWP/268/95), and more preferably greater than 5 log1 () or further greater than 6 log1 () ' Therefore, it is unlikely to cause a blood-borne infection of the lipid envelope virus when transfused to a patient. The condensable blood platelet growth factor concentrate obtained by the method of the present invention or the method of the present invention further comprises the following functional growth factors: platelet-derived growth factor (PDGF), which is a heterodimer of A and B chains and. / or homodimer of AA and / or BB chain (PDGF-AA, PDGF-AB, PDGF-BB); Transgenic growth factor (TGF-β) superfamily, which specifically contains TGF-βΙ and/or TGF -P2 and/or bone morphogenetic protein (BMP), insulin-like growth factor (IGF); epidermal growth factor (EGF); connective tissue growth factor (CTGF); primary fibroblast growth factor ( bFGF) and vascular endothelial growth factor (VEGF). In a special embodiment, the condensable platelet of the present invention has a PDGF concentration of 15 1344963 long-factor thick solution higher than 9 ng/ml thick solution, and is preferably about 100 ng/ml thick liquid, more Preferably, it is higher than 12〇ng/ml thick liquid, and more preferably higher than 150 ng/ml thick liquid, more preferably higher than 180 ng/ml thick liquid, and even more preferably higher than 25 tons Thick liquid 0 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 叩 / 1111 thick Liquid, more preferably higher than 16〇11§/1111

The thick liquid is more preferably a concentrated liquid higher than 180 ng/ml, and even more preferably a thick liquid higher than 250 ng/ml. In a particular embodiment, the concentration of IGF in the clotting platelet growth factor concentrate of the present invention is at least similar to that of the starting platelet concentrate and is preferably greater than 65 ng/ml thick, more preferably It is more than 75 ng/ml thick liquid, more preferably higher than 8 ng/ml thick liquid, and even more preferably higher than 100 ng/ml thick liquid.

In a special embodiment, the ECT rich in the clotting platelet long-factor thick solution of the present invention is higher than the G.5 ng/ml thick liquid, preferably higher than lng/ml thick liquid, more preferably high. It is preferably more than 2 ng/ml thick liquid, and more preferably more than 25 ng/ml thick liquid, at 15 ton / as a dope. In a particular embodiment, the condensable platelet growth factor concentrate of the present invention or obtained by the method of the present invention is suitable for use in a condition of lipid content. And the phrase "a condition requiring a low lipid content" used in the present invention means that the condensable liquid growth factor of the present invention or the method of the present invention has been removed from the lipid (depleted Xun) The amount of 'cholesterol, triglyceride, hdl and its initial platelet thick concentrate in the condensable blood 16 3 : T thick solution obtained by the present invention or by the method of the present invention is extremely low in quality. The lipid in the thick platelet growth factor concentrate is used for the therapeutic use of the sputum, for example, the risk of the disease (4), or the amount of the risk for high heart risk = salty, ': Patients who have received treatment offer extremely low levels " greatly reduce the risk of vascular occlusion caused by the formation of fatty plaques (Plaq=) in the arterial and venous systems. The invention may be obtained by the method of the invention or by the method of the invention. Condensed thick liquid towel, the amount of bile rotation is less than (10) g coagulable, sputum platelet growth factor thick liquid, preferably less than 5 〇 - condensed ===: liquid and more preferably lower than the knot Birth! Can condense OOmg/dl clotting platelet growth factor = : = 5 〇mg/dl condensable platelet growth 乂 preferably less than 3 - condensate..., plate = 、, night and better for the low condensable mg/cU condensable μ plate Raw (four) sub-thick liquid, less than 30 mg / dl clotting platelet growth factor thick liquid, and 〗 〖= 15 10mg / d condensable platelet growth factor thick liquid. More box is lower than better, in The present invention or the platelet-derived (four) sub-rich coffee 17 1344963 mg / dl clotting platelet growth factor thick solution, preferably less than % mg / dl clotting platelet growth factor thick liquid, more preferably low a platelet growth factor thick solution which is condensable at 2 mg/dl, and more preferably a platelet growth factor thick solution which is less than 5 mg/dl. Further, the present invention or the method obtained by the method of the present invention The quality of the lipid in the condensed platelet growth factor thick solution is very low and/or undetectable, so it has better stability. When the platelet growth factor is purified on a large scale, the method is easier to carry out. In the present invention, or by the method of the present invention The clotting platelet growth factor thick solution obtained by the method does not cause blood cell-related transfusion reaction. "Blood cell-related transfusion reaction" means that the clotting growth factor thick liquid does not have intact living cells (such as red blood cells). , platelets and white blood cells), so it can avoid causing a known range of known transfusion reactions in patients, including immunization (such as alloimmunizaticm) 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 will develop 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 types: ("type 1 HLA, platelet and white blood cell co-owners; (7) type 2 HLA, in some white blood cells; (3) granular white a ball specific antigen (4) platelet-specific antigens (for example, human platelet antigens ;ρΑ); and (5) red blood cell-specific antigens. More specifically, 'in the preparation method of the present invention, live blood is fine, (red blood cells, white blood cells and Platelets) will be destroyed/dissolved by solvent_cleaner, thereby reducing (and better preventing) patients exposed to foreign antigen 1344963 to u, plate 3 mu method can be obtained from patients: small plate: condensed blood two The growth factor is different from the present invention. The invention may be further selected from the method of the present invention to be further contained from the sorghum I long thick liquid to the sensitizing protein and/or the π (the suspected worm) transparent adhesion Protein, thrombin sensitivity VIII ^ IX ^ (P-thrombin) ) V. VII. The group of eggs from the coagulation factor and the composition of the Willer's factor, the clotting platelets of the present invention The raw material of the raw (four) even egg (four) is higher than 〇 2 g / L / Chen sputum, and more preferably about 3 g / L thick liquid. · Growth plate source rc is better than.. a thick liquid: and better = other embodiments, the condensable of the present invention Platelet growth factor: a vm clotting factor and/or a van derrick factor 〇.9 IU/mU auxac thick liquid in a thick liquid, and more preferably about 丨m/ml thick liquid is in another embodiment The sample towel, the curable === thick liquid step obtained by the method of the invention comprises immunoglobulin, as in the special embodiment, the immunoglobulin IgG in the clotting platelet growth factor thick solution of the invention The concentration is preferably greater than 5 g/L of concentrated liquid, and more preferably about 1 〇g/L of concentrated liquid. In another embodiment, the present invention or condensable by the method of the present invention 19 1344963 The platelet growth factor concentrate 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), Skin growth inhibition. Agent (EG) 〇, keratinocyte 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), tissue limb release factor, human collagenase inhibitor, platelet antibacterial protein-1 (PMP-1), thrombin-induced platelet antibacterial protein-1 (t-PMP) , thrombin peptide 1 (throinbocidin-1, TC1) and thrombin-2 (TC2). In another embodiment, the condensable platelet growth factor concentrate obtained by the present invention or by the method of the present invention further comprises at least one protein selected from the group consisting of serotonin, cathepsin (cathepsin), albumin, platelet basic protein _pBp (CXCL7), neutrophil activating protein 2 and _4 (NAP-2, -4), _ somatostatin (somat〇statin 'SST), RANTES , CTAP-3, placental protein 14 (PP14), SCUBE annexin U (heatinensis) i - heat shock protein 27 (HSP27), and heat shock protein 60 (HSP60). • In a particular embodiment, the albumin concentration in the condensable platelet growth factor concentrate of the present invention is preferably higher than 3 〇 g/L thick. In another embodiment, the indestructible platelet growth factor concentrate of the present invention or by 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 metalloproteinase MMP-b-2, -9, -13, autocrine ERK (ext.Ceil reg. kinase), autocrine and paracrine protein C (PC), and trace enzymes such as aldolase, carboxyl peptide, acid phosphatase, arylsulphatase, β-galactosidase, β-gluconate acidase, --glycerophospho-acidase (P_glycer〇lph〇sphatase), oc/β-glucosidase, α/β-fucosylase (a/p_fUc〇sidase), α-mannosidase (α- Mannosidase and α-arabinosidase ° 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, α1 -α2 antitrypsin, α2-antiplasmin, α2_ giant ball Protein, ci-INH, induced sputum-2, -6, -7 (TGF-β superfamily), ECM remodeling factor (ECM remodelling factor 'induced sputum, TNF-α, elastase..... .), autosomal and paralytic lysophosphatidic acid (LPA), HMGB1 (amphibiegulin), ATP, ADP, GPT, GDP, Ca2+, Mg2+ and/or Zn2+. Another object of the invention is a pharmaceutical product comprising the condensable platelet growth factor concentrate of the invention or obtained by the method of the invention. "Medical products" means plateiet gei, platelet glue, growth factor-rich fibrin glue and/or sealant, artificial eagle. Another object of the present invention is the use of the coagulated platelet growth factor of the present invention or obtained by the method of the present invention in a medium, wherein the medium is suitable for use in fibroblasts, chondrocytes, osteoblasts, keratinocytes, In vitro or in vivo culture of stem cells and/or transplanted cells. Another object of the present invention, a cell, a chondrocyte, an osteoblast, a horn, is suitable for in vitro or in vivo study of fibroblasts, a stem cell, and/or a transplant invention or by the present invention; This factor. The condensable platelet growth of the female part of the small plate thick liquid Lin Nui Cai / value and in from 2. . Up to 5^^^^ minutes to 6 hours, more preferably at a temperature of from 25 ϋC _ ^; and removal of the aforementioned solvent and/or cleaning agent by alcohol extraction and/or stratification. In a preferred embodiment, the culture is carried out for a time ranging from 2 to 4 hours, which is carried out at a physiological 1) 11 value, such as 1 > 11 in the range from pH 7 ΡΗ to ΡΗ 7·5 ( When the starting platelets are fresh platelets, or at a value ranging from 6.8 6.8 to 8.2 (when the initial blood small f is expired and/or cold/dong small plate). Advantageously, the culture temperature is about 31 °C. Suitable solvents for use in the process of the invention are di- or tri-alkanes, dish esters such as tris-(η-butyl)phosphate, tris-(t-butyl)phosphate, ~hexyl) Filler, tris(2-ethylhexyl)phosphate, tris(n-mercapto)phosphonium, di-(n-butyl) acid vinegar, di-(t-butyl) dish warm vinegar, Di-(n-hexyl)phosphate, di-(2-ethylhexyl)phosphate, di-(Iinyl)phosphoric acid and dialkyl phosphates having different alkyl chains. Di- or trialkyl phosphates having different alkyl groups may be used, such as bis-(II-butyl)phosphoric acid. Particularly preferred is a trialkyl phosphate which is tris-(n-butyl)phosphate 22 (ΤηΒΡ). In the present invention, a suitable cleaning agent for the use of a polyoxyethylene derivative of the acid = fat屯Γτ 0 The partial ester of sorbitol (for example, the name iween 80 丨 / , βΛχ Γ 、 , also known as "polysorbate 14 ii Aweel20 j ^ ^ #) (^ $ "τ .Oral ethoxylated alkyl phenol: "Triton Χ-100" or Iriton X-4S, 仏^ _"). Further consideration of the cleaning agent is sodium deoxycholate. For example, N-dodecyl-n,n-dimercapto-2-l-hexan-N,N-dimethyl-2-ammonio-l-ethane SUlph〇nate. "Triton X-45", "Triton X-100" and "Tween 80". In a special embodiment of the present invention, the starting platelet thick liquid is co-cultured with an agent and a detergent, preferably TnBp and THt〇n X-45 are co-cultured. & Advantageously, the final concentration of the solvent or the aforementioned cleaning agent, or the solvent and the individual final concentration of the aforementioned cleaning agent are contained in a volume of from 0.2 to 2. More It is included in the range from G 2 to 2% by volume = which is based on the volume of the aforementioned platelet thick liquid. In the -1 parent implementation, 'the aforementioned initial platelet thick liquid system is co-cultured with 2% ΤηΒΡ In another preferred embodiment, the starting platelet thick liquor is co-cultured with 1% ΤηΒΡ and 1% Triton χ_45. ^ The aforementioned solvent and/or detergent can be extracted by oil with a pharmaceutical grade oil and The financial method (such as f-column chromatography)* is extracted from the biological fluid, so that the solvent and/or detergent in the thick liquid has been removed. The aforementioned pharmaceutical grade oil may be a natural oil (for example, from plants or animals). An extracted oil) or a structurally similar synthetic compound. Suitable natural 23 1344963 oils include castor oil (or rjcinus 〇 ii), soybean, sunflower, sunflower oil, cottonseed oil. Preferred synthetic compounds are synthetic triacids. Examples of suitable synthetic triglycerides include triolein, tristearin, tripartre = (tripgf!?ltln), trimyristin, and combinations thereof. Medicine, medicine, etc. The amount of oil to be extracted at least 8 square lipid soluble process chemicals%, (process chemical) in the amount of, the fuel consumption of from 2 to &

The % is reset, which is calculated as the weight of the lysate of the platelet thick solution; preferably from 5 to 15% by weight, and more preferably from 5 to 5% by weight. The oil extraction can be carried out once, preferably twice, and the second is particularly dependent on the concentration of the oil. Soil ‘, ''—The aforementioned solvents and/or cleaners can also be removed from the blood plate, thick liquid lysate by column chromatography. Column chromatography can also be carried out after oil extraction or directly after solvent and/or detergent treatment.

Suitable chromatographic columns include reversed phase (hydrophobic interaction), shellfish, or protein-adsorbing matrices such as ion exchange (anionic matrix and affinity (eg, immunological affinity or immobilized heparin & ruler, size-exclusion) Base. The preferred reverse phase substrate is CU cerium oxide filling material, SDR (solvent_cleaner removal) hyper D (Pall corporation), polystyrene absorbent () and

Ambedyte ( Rohm ). Even though the c! 8 series is generally preferred for industrial scale chromatography, the tC18 ceria loading material is also considered. 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_Sephadex A-50 ^ DEAE-Sepharose FF ^ Q-Sepharose > DEAE-24

Toyopearl 650M, DEAE-Hyper D. These adsorbing substances are used for binding to growth factors 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 20 ppm, and even more preferably less than 1 〇 ppm, less than 5 Ppm, and 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, More preferably less than 50 ppm, and even more preferably less than 1 〇 ppm °. After removing the solvent and/or detergent by oil extraction and/or chromatography, a further step can be added to make the non-sleeve The virus (such as the small virus B19, or possibly the hepatitis A virus (HAV)) is deactivated or removed, such as nanofiltration, and more particularly 75_nm, 35_nm, 2〇-nm, l5_nm or l〇 Nanofiltration was carried out using a -nm pore size filter. In another preferred embodiment, a centrifugation step is performed after oil extraction and/or chromatography to remove cellular debris. Advantageously, the centrifugation step is carried out at 800 to 20,000 x g for 3 minutes, and preferably at 10,000 x for 15 minutes. In another preferred embodiment, a filtration removal step is performed after oil extraction and/or chromatography to remove cell debris. Advantageously, the aforementioned passage step is carried out with a transitioner that gradually changes from 1 μηη i 〇 2 μηη, which also removes bacteria. In a particular embodiment, the platelet thickening liquid used as a starting material in the method of the present invention is a single or mixed standard gold plate thick liquid, for example, a platelet thick liquid for blood transfusion use. Blood 25 1344963 Small plate thick liquid can also be obtained from the whole blood buffy brown layer separation method. One unit of platelet concentrate derived from the buffy coat is typically 30 to 50 ml. Platelets derived from buffy coats can be used as starting materials, which are a single unit or a mixture of multiple single years, such as when forming a therapeutic unit, which is a mixture of 4 to 6 early and early positions (eg, Guide to the preparation, use and quality assurance of blood components - 13th edition (2007), edited by the Council of Europe

Platelet concentrate can be obtained by blood separation (referred to as platelet preparation by whole blood donation), cytapheresis or plateletpheresis (see, for example, Guideto the preparation, use and quality assurance of blood components). , 13th edition (2007), edited by the Council of Europe), and by using MCS+ (Haemonetics),

From 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 3 〇〇 mi platelet concentrate) from each donor.

In a preferred embodiment, the method of the present invention comprises a preliminary step of preparing a starting plate thick liquor. Advantageously, methods for preparing a starting platelet concentrate include, but are not limited to, standard blood bank procedures (eg, small blood = separation or platelet preparation via whole blood donation) and focus on private procedures (eg, using blood cell preservatives / Separator or table device f 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 or for a few weeks. In a particular embodiment, the platelet concentrate used as the starting material 26 in the method of the present invention may further comprise white blood cells and/or red blood cells. Therefore, the starting platelet thick liquid It may contain several differentiated and unactivated white blood cells, such as lymphocytes, neutrophilic granulocytes, and spheroids. In particular, neutrophils and mononuclear spheres are rich in bone marrow peroxidase ( a particle of myeloperoxidase) which catalyzes the oxidation of chloride to form hypochlorous acid and a derivative of its reactive oxygen species, wherein the aforementioned reactive oxygen derivative is Bacteria oxidant 'toxic to microorganisms and fungi.

Thus, when the starting platelet concentrate comprises several differentiated and non-activated white blood cells, the condensable platelet growth factor concentrate obtained by the method of the present invention further comprises an antimicrobial component derived from white blood cells. In a preferred embodiment, the white blood cells in the platelet concentrate used as the starting material are eliminated, more preferably by leukoreduction, to avoid the occurrence of proteases and acid hydrolases in the white blood cells. Promotes an inflammatory response. Leukocyte removal also reduces the risk of prion contamination. _曰

A healthy person's normal platelet count typically contains between 150,000 and 400,000 platelets per mm3 of blood, i.e., 15 to 4 X X 〇 9 platelets/L. This "normal" platelet count is about 5% of healthy people, and the remaining 5% may be statistically abnormal. The blood plate is § ten (very low or very high). When platelets are collected by platelet separation, the platelet count in a bag of 25 〇ml is generally higher than 1.2 X 1 〇 9 platelets per ml ' 'the plate count is equivalent to more per bag (unit) About 3 x 1〇n small plates. In the preferred embodiment, the number of blood platelets in the initial, plate thick liquid is 3 to 10 times higher than the number normally seen in blood. Another object of the present invention is a method of forming a blood clot, which comprises the steps of: mixing the condensable platelet growth factor thick solution obtained by the present invention or by the method of the present invention with thrombin, or A mixture of activators of a blood agglutination chain reaction, such as activated FVH, or bovine or human thromboplastin, a condensable platelet growth factor concentrate obtained by the present invention or by the method of the present invention Prior to the formation of a blood clot, an antifibrinolytic agent may be added to the condensable platelet growth factor concentrate obtained by the method of the present invention or the method of the present invention to inhibit or slow down fibrin; natural proteolysis in glutathione Enzymes such as plasmin break down fibrin clots. In a particular embodiment, the antifibrinolytic agent is aprotinin, but the concentration is > 1 〇mg/ml of tranexamic acid or 8 •• aminocaproic acid (epsilon aminocaproic) Acid) can also be used as an alternative compound to trypsin. In the liquid form, the concentration of the inhibitor peptide is usually 3000 KIU or less. After mixing the clotting platelet growth factor concentrate and thrombin components, the final degree of the pancreatic peptide is typically half that of the starting solution. The condensable platelet growth factor thick solution obtained by the present invention or by the method of the present invention may be further mixed with an artificial scaffold (for example, a manufacturer of collagen egg=, chitosan, ceramics), or Know the self-blooded fibrin glue or fibrin sealant. Additional compounds may also be added to the condensable blood L-plate 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. The condensable blood 28 plate growth factor> agricultural thick liquid obtained by the present invention or by the method of the present invention and the suspected blood enzyme mixed in the __ will reproduce the final step of the blood agglutination chain reaction, and (4) the protein progenitor gradually Polymerization forms a blood clot or insoluble fibrin. The term "thrombin" as used in the present invention relates to thrombin which can be obtained from a source, and preferably refers to bovine thrombin; and if it is used for therapeutic use in humans, it is more preferably human thrombin. A person activated by CaC12, plasma or python thrombin (batr〇x〇bin, another fibrinogen j blood protein transfer, from the snake to add the heart of the $10 (10) to the snakes) Activated FVII, or human or bovine thromboplastin can also be used as a replacement for thrombin. In a preferred embodiment, thrombin to volume is added to one volume of the condensable platelet growth factor concentrate of the present invention or obtained by the method of the present invention. In a preferred embodiment, the thrombin concentration ranges from 2 〇 IU/ml to 1 〇〇〇 iu/m 卜, and the final concentration of thrombin is about 500 IU/ml to ensure continuous and rapid. The fibrinogen polymerization will result in soluble fibrin, followed by stable (insoluble) fibrin, or when the specific surgery is desired to slow the polymerization, the final concentration will be lower. The two components of about 4 to 25 IU/m (i.e., the clotting platelet growth factor concentrate and thrombin obtained by the method of the present invention or by the method of the present invention) can be administered sequentially or simultaneously by a double syringe system. Apply at the location to be repaired, with or without the use of an endoscopic delivery device, and using spray or absorbable sponges (see Radosevich et al., Fibrin sealant · Scientific Rationale, Production Methods ,

Properties, and Current Clinical use,,, Vox Sanguinis, 1997, 72: 133-143 and Marx G, "Evolution of fibrin glue 29 1344963 applicators", Transfus Med Rev, 2003; 17(4): 287-98. The method of application, the above is incorporated herein by reference. - Therefore, another object of the present invention is the use of the present invention or by the method of the present invention; the use of a spinnated platelet growth factor thick solution for therapeutic applications, and the use of a blood clot to form, or in vivo External or . Cell culture use for in vivo studies. When it is used for cell culture in vitro or in vivo studies, the clotting platelet growth factor concentrate obtained by the present invention or by the method of the present invention may be present in the medium in an amount ranging from 1% to 3%. 'More preferably from 2% to 20%, and even more preferably from 3% to 10%, based on the volume of the medium. The primary therapeutic applications of the aforementioned thick liquid/thrombin mixture include, but are not limited to, dental surgery, implantation, orthopedics and oral surgery, hand surgery, soft tissue and hard tissue healing and reconstruction, cardiovascular, thoracic, Or gastrointestinal surgery, neurosurgery, general surgery/trauma surgery, ophthalmology, otolaryngology, urology, and surgery in patients with anticoagulation or coagulopathy. The above and other objects, features, and advantages of the present invention will become more apparent from the description and appended claims. EXAMPLES Materials and Methods 1.1 - Collection of apheresis platelet The initial platelet thick solution (PC) was collected by a volunteer donor using the MCS+ multi-component system (Haemonetics, Braintree, USA). The whole blood is obtained through an intravenous catheter, using intermittent flow and anticoagulant (lml anticoagulant dextroglucose solution formula _ per 〇mi blood). The gray plate-rich plasma 30 1344963 (PRP) is automatically separated from other blood components by centrifugation and collected in a sterile, single-use disposable bag, and the red blood cells and the plasma will return to the donor. in vivo. 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 counts Platelets, white blood cells (WBC) and red blood cell counts use cells

Counter (ABC Vet Automatic Blood Counter, ABX

Diagnostics, France) for the determination. 1.3- Treatment of starting platelet thick solution α-research design

The starting platelet thick liquor was processed according to the study design of the first panel. Briefly, the same donor starting platelet concentrate (3 〇〇 ml) was gently mixed and divided into two equal volumes of pool (15 〇 ml). Subset Pool 1 is directly subjected to S/D processing and is not activated first. When the subset pool 2 is in the presence of 23 mM CaCl 2 and glass beads, it is activated under conditions that cause platelet gel formation, as described below; the resulting release is carefully and carefully recovered by pipetting (average Volume is equivalent to 120

Ml' was lost in the platelet gel due to 5 3 〇 ml) and then subjected to S/D ' Ϊί extraction. Among the samples obtained from the starting platelet thick solution, there were unactivated platelet sputum after S/D treatment, = subset pool 2 after treatment, and treated activator 31 b-platelet activation: In the presence of glass pellets, 1 MCaCl2 (Sigma, lot number k56k0688) was added to the platelet concentrate (subset pool 2) for/tongueization with a final degree of 23 mM. The mixture is gently spun and mixed until the gold clot forms, which typically takes place in 5 to 8 minutes. The mixture was allowed to reactivate for a further 6 minutes, under which conditions the optimal platelet-derived growth factor was released. The supernatant was poured out' 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 finalize The concentration (v/v) was 1% ΤηΒΡ 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 gentle agitation. Upon completion of the S/D treatment, soybean oil (Sigma, Missouri, USA) was added to the S/D-platelet subset pool to give 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 ΤηΒΡ and Triton 32 1344963 X-45 to less than 10 and 100 ppm, respectively. 1.4-Effects of centrifugation and lysing agent types In order to study the effect 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 A pool of 150 ml subsets. One of the subset pools was subjected to high speed (10000 X g) centrifugation to make the platelets a pellet and the supernatant was treated with 1% φ TnBP-1% Triton X-45. Another uncentrifuged subset pool was subdivided into two 75 ml subset pools, one of which received 1% ΤιιΒΡ-1% TritonX-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 Experiment In order to rule out the fact that CaCl2 activation does not completely activate platelets, two platelet concentrates (14 ml) were activated as described above in the presence of 0.23 M CaCb and glass spherules. After φ is gently mixed and vortexed at room temperature for 60 minutes, l〇ml of a small plate thick liquid release is recovered, and 0.5 ml of 1000 international units (IU) _ /ml of bovine thrombin (Thrombin_JMI, 52604-7102- 1, J〇nes

Pharma, Saint-Louis, MO) to a final concentration of about 48 IU/ml. The mixture was gently mixed and spun at room temperature for 60 minutes. Thereafter, l°/〇TnBP-l% Triton X-45 treatment and oil extraction were carried out. In a parallel experiment, two platelet concentrate samples (10 ml) were also directly activated with the same bovine thrombin of 0.5 meg, and gelled in a few seconds, followed by gentle spin mixing for 60 minutes. Samples were taken at various steps in the experimental procedure's centrifugation at 10,000 xg and frozen at _8 〇 °C until blood platelet 33 1344963 plate source growth factor analysis. 1.6-Growth Factor Determination Samples were taken at each step of the procedure. Centrifuge at 1 〇〇卯 X g for 15 minutes (Microfuge® 22R, Beckman c〇ulter

Fullerton, CA), makes platelets and/or cell debris a mass, and delivers cell-free supernatant for platelet-derived growth factor measurements. In addition, a parallel experiment of 15 minutes was also performed with a centrifugal force of 800 xg. Immediately thereafter, the supernatant was cold-bound at -8 CTC. 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 times with Calibrator diluent (RD6-11). The wells were incubated for 2 hours, washed, and co-cultured with the enzyme-bound PDGF-AB antibody for 3 hours at room temperature. The cleaning buffer (Wash Buffer;) was used to clean the wells' and then the Substrate Solution was added at room temperature for 20-30 minutes. The hole is protected from light. A Stop Solution was added to each well and the absorbance at 450 nm was measured using a microtiter plate reader. The minimum detectable dose is 1.7 pg/m b b-TGF-βΙ

TGF-βΙ was determined using a Quantikine ELISA kit (DB100B, R&D 34 1344963 SYSTEMS). The sample was diluted 1 fold 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 96-well microtiter plate coated with TGF. Prior to TGF-βΙ analysis, acid activation and neutralization reactions were performed to activate latent TGF-β 1 to an immunologically active state. For this purpose 'mix 0.5 ml sample with 〇.1 m丨 in HC1' for 10 minutes at room temperature, add 0.1 ml of i 2NNaOH / 0.5M HEPES (N-[2-hydroxyethyl] Ν0·[2-ethanesulfonic acid]) (Sigma 'Η-7523) was neutralized and centrifuged. The total TGF-βΙ content of the supernatant fraction was then determined. Aliquots (5 μl) were added to the microtiter plate in duplicate, then capped and incubated for 2 hours at room temperature. Thereafter, the wells were conjugated with TGF-βΙ conjugated to the enzyme, and cultured at room temperature for 1.5 hours. The measurement is completed 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 SYSTEMS, Minneapolis, MN). The sample was diluted 20-fold with Calibrator diluent (RJD6N). Add 200 μΐ of the standard, control or sample to the well. The wells were incubated for 2 hours at room temperature. Each well was pumped' and filled with 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) measured 35 1344963 at 450 nm in 30 minutes. The minimum detectable dose is 0.7 pg/πύ. d-IGF-1 IGF-1 was quantified using a Quantikine ELISA kit (DG100 from R&d SYSTEMS). The sample was diluted 1〇〇 with Calibrator Diluent ‘ (RD5-22). As stated by the manufacturer, the minimum detectable dose range is from 〇.(8)7 to 0.056 ng/m and the mean MDD is 0.026 ng/ml. 150 μM of the measurement diluent φ (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 conjugated 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 two-tailed paired Student test. The p value is small • The 〇.〇5 series is used to estimate the difference in the average PGF concentration in the different steps of the platelet preparation procedure (the Significance 〇f the - differences). The value is expressed as inconspicuous (NS, < 〇.〇5), <0.01 or <0.00, and when the value is close to 0.05, a true p value is displayed. The preparation and separation of samples comparing I.8-PC, S/D-PC and Act-PC correspond to the initial gold plate thick solution (PC), solvent/cleaning, (1%1^ and 1%Trit). 〇nX_45) The treated initial platelet concentrate (S/D-PC) and the CaCl2-activated starting platelet concentrate (ACt_PC) were separated by SDS-PAGE to compare the 36 1344963 protein content. • Mix 20 pg of each sample with 5 plNuPAGELDS sample buffer. (4x ) ( Invitrogen ), 2 μΐ NuPAGE reducing agent (ι) (Invitrogen) and deionized water to give a final volume of 2 μμΐ (activated) The sample of platelet concentrate was 21 μΐ). The resulting mixture was then heated at 70 ° C for 10 minutes and subjected to SDS_PAGE using 4-12% polyacrylamide gradient gel (NuPAGE Bis-Tris, Invitrogen). The φ protein separation was carried out for 35 minutes under a constant voltage of 200 V, wherein the predetermined current was 150 mA/gel. The resulting gel was stained with Coomassie blue R-250. The protein marker ^1 & also 12 unstained standard (11^Du Honghong 11) is used to measure the molecular weight of the protein in the sample. The Mark 12 marker was loaded onto a NuPAGE novex 4-12% Bis-Tris gel (Invitrogen) with MES and separated by Coomassie Blue R_25® after separation. The corresponding results are disclosed in the fourth figure. The egg white map (pr〇file) of the initial platelet concentrate treated with the solvent/detergent method showed no significant difference from the untreated initial platelet concentrate, whereas the activated platelet desert solution The protein map - the spectrum is significantly different, the bands in the 40 to 70 coffee area disappear, and these bands correspond to the alpha, P and subunits of fibrinogen, 63.5, 56 and 47 respectively. kDa. 1.9-Growth factor activity assay In order to determine whether the growth factor obtained by platelet S/D treatment (1% TnBp_1% Tdt〇n X-45) can maintain activity, human osteoblast-like MG-63 cell line was used for in vitro cells. Cultivate research. 37 1344963 MG-63 cells were treated with growth factors obtained from S/D-PC or from Act-PC • concentrated, 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-glutamic acid, Eagle's BSS (Balanced Salt Solution) ), adjusted to 1.5 g / L sodium bicarbonate, 0.1 mM non-essential amino acid, 1.0 mM sodium pyruvate, 10% φ heated 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). After 37 min of C 作用C, the cells were detached, and 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-diphenyltetrazolium bromide (MTT) assay. Electron microscopy was also performed to study cell morphology. • Use of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-continuous phenyl)-2H-tetrasporin (MTS) Assays to monitor cell proliferation. Briefly, 500 μM of MTS (Celliter 96 Promega Corp., USA) was added to each sample. After incubation at 37 ° C for 60 minutes, the absorbance at 490 nm was measured using a microplate reader. 1.10-Virus Deactivation Assay The solvent potency study for solvent and/or detergent treatments used to initiate platelet thick lysis is performed on a lower scale experiment based on international benchmarks such as EMEA and WHO recommendations. get on. 38 (50 ml of the initial blood subtype of immunodeficiency virus) and ^^, ^ related virus (5) V, human toxicity of the abdominal virus (BVDf) widely stored 7 liquid · bovine rabies virus (PRV); Hepatitis virus model; when used, sometimes used = there are other convenient in vitro model viruses (VSV). These viruses were stored in a concentrated solution of vesicular stomatitis virus plate and added to the stock suspension to introduce a mixture of the initial blood virulence virus _f f ^ % tdtGn X_45. The time points were estimated, one/month of swimming " 丨 before treatment and during treatment during different in vitro cell culture; kinetics of toxicity. Value is used to indicate. The data obtained from the data are within TCIDw/ml so that the four disease controllers do not, and the S/D treatment will be activated 5 minutes after the treatment (four), and the mother will be completely deactivated (non-toxic infection force. 丄' two is 6.4 log10, and The vsv is >7 〇1〇. Therefore, the solution = $ lysate will ensure that the lipid envelope virus may be present in the initial blood sputum cell product when the t strong virus deactivates the drought 1.11 expires frozen Platelet growth factor composition, the platelet thick solution which was expired (more than 5 days after collection) was transferred to the c library for one month, and then they were decomposed in 3rc water and subjected to the same experiment as the fresh platelets described above. Factor content, the corresponding results are revealed in the 1.12 - gray reduction block formation assay from the S / D treatment of the initial platelet thick solution derived from the growth factor 39 thick liquid is extracted by oil to remove the solvent and detergent Recycling was carried out. 5 ml of the resulting growth factor concentrate was introduced into a 5-ml syringe. 5 ml of 500 IU/ml bovine thrombin was introduced into another 5-ml syringe. Double syringe feeder connected to the nozzle. Let these two The components pass through. The platelet gel will form within 5 seconds. II. Results II. 1-cell counts Ten platelet separation concentrates from ten different donors were studied. Platelets from platelet separation The results of the concentrated solution averaged 1064 ± m2 X 106 platelets/mi (range: 782_ 1358 X 106 platelets/ml), and the average WBC count was 0.1125 ± 0.025 X 10 cells/ml (pillow circumference 0.0-1.5 ) 'and average RBC content is 0.0212 ± 0.025 X 1 〇 6 / m 卜 II. 2 - growth factor content PDGF-AB, TGF-P1, EGF and coffee] average concentration in standard platelet preparation ± standard deviation (SD), minimum The maximum value and the p value are shown in Table 2. The individual data points obtained from each series of experiments can be found in the second figure.

PDGF-AB TGF-βΙ EGF IGF (N=10) (N=10) (N 2 8) (N = 6) m 13.8 16.6 <0.0007 83.4 A SD 14.3 14.3 - 32.8 Min. 2.2 1.1 60.1 Max. 49.5 36.0 - 162.7 m 184.4 192.2 2.2 88.4 B SD 80.2 37.4 1.6 33.5 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 C SD 35.5 14.1 0.6 34.9 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 D SD 45.9 27.2 1.0 39.7 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 B vs C < 0.001 < 0.001 0.044 < 0.001 B vs D < 0.001 < 0.001 0.087 < 0.01 CvsD NS NS NS NS Table 1: In the starting platelets (A) at 1% TnBP-l %TritonX-45 1344963 After treatment (B), and after CaCl2 activation (C), the release compound was subjected to 1% TnBP-1% Triton X-45 treatment (D) for the average growth factor concentration (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-TritonX-45, its content increased significantly (p<0.001) to 184,4 ± 80.2 ng/ml, which is equivalent to approximately 13-fold increase over the initial platelet concentrate. When the initial platelet concentrate is first activated by CaCl2, its content 41 will also increase significantly (84.6 ± 35.5, p < 〇.〇〇l) 'but the increase is less (about 6 times increase), and it is in the subsequent S The /D process remains substantially unchanged (88.3 ± 45.9, NS). In the unactivated treated platelets, the PGDF-AB content was significantly higher than that in the activated, and activated/S/D treated platelets. Similar data were also found for TGF-βΙ. The average TGF-βΙ content in the starting platelet concentrate was 16,6 ± 143 ng/= (N=10). After direct S/D treatment, the TGF_pi content will increase by nearly 12 times that of the starting pc' to 192.2 ± 37.4 ng/ml (p<〇.〇〇1). When the initial platelet concentrate is first activated by CaCl2, the content is increased by ^ 4 ^ (63.8 ± 14.1 ng/ml) (p < 〇.001) and remains in a distinctly different state during the subsequent S/D treatment period ( 68.6 ± 27.2 ng / ml). Also: In the S/D-treated platelets, the average TGF_pi content was significantly higher than that in the activated, and activated, S/D-treated platelets (p<0.001). In the initial platelet thick solution, pg/ml could not be detected, but it became detectable after S/D treatment, and the average content (2.2±1.6 ng/ml ' .6) was significantly higher than (p&lt ;〇〇5) The value obtained after CaCl2 activation (0.9 ±0.6 ng/ml) (p<〇〇5), or significantly higher than the value obtained after SiO2 activation followed by s/D treatment (丨4 + 1.0 ng) /ml) The average level of IGF-1 in the starting platelet concentrate was 83 4 + 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 ± phantom 5, p = 0.025). In the preparation of platelets after CaCh activation, its average content was even slightly higher (P^.OOl) (ll7.2±34 9 ng/ml) and remained stable after subsequent S/D treatment (H2.4 ± 39.7 ng/ml). 42 1344963 The total amount of PDGF-AB, TGF-β, EGF and IGF-1 returned by direct S/D treatment (153 - ml) in 150 ml platelet concentrate was 28213, 29406, 336 and 13525 ng, respectively. After CaCl2 was activated, it was 1〇152, 7150, 1〇8 and 14064 ng, respectively; the 20% average volume loss caused by platelet gel formation (120 ml) was also counted. This does not allow the S/D treatment to be more effective in releasing pDGF-AB, TGF-βΙ and EGF than CaCl2 activation. Table 2 compares platelet-derived growth factor in the initial fresh platelet concentrate (A), after centrifugation (centrifugation into a mass and removal of platelets), when the supernatant is subjected to TnBP-Tdton X-45 treatment (9) And the content of the initial platelet thick solution after S/D treatment (C) or S/D treatment (D) with 2% TnBP at 1% ΤηΒΡ-1% Triton X-45. The levels of PDGF-AB, TGF-β, EGF and IGF-1 in untreated platelet-thick liquor (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; p<〇.〇〇) l) 'The content of IGIM is high, but similar to the amount in φ starting platelet concentrate (75·4 vs. 72.; 2 ng/ml). Therefore, the release of PDGF_AB, TGF-βΙ and EGF during S/D treatment depends on the presence of gold platelets. The increase in 'platelet-derived growth factor' content in platelet lysates treated with 1% TnBP-1% Triton X-45 or 2% ΤηΒΡ was similar. Further, after the platelet concentrate is treated with 1% tritoiiX-45 or 1% tritonX-100, the subsequent detergent treatment is removed by tC18 adsorption (SPlT45tC18 and SPlT100tC18) without oil extraction; The growth factors PDGF-AB, TGF-βΙ, IGF and EGF observed after treatment with 2% ΤηΒΡ or 1% tonηΒΡ and 1% Triton Χ-45 were in a range of 43 1344963, indicating that platelet dissolution The extent of the cells is substantially the same as in the case of using such a solvent and detergent combination or a solvent or detergent alone. Finally, the data (not shown) indicate that centrifugation of the sample at xg or 800 Xg does not affect the amount of platelet-derived growth factor measured in this assay. PDGF-AB TGF-P1 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. Various growth factors in the starting platelet concentrate (a) The centrifuged platelet-free supernatant is treated with TnBP-Triton X-45 (B), after TnBP-Triton X-45 treatment (C) or after 2% ΤηΒΡ treatment (D) Concentration of concentrated liquid + (ng/ml). Further, the concentrations of fibrinogen, fibronectin, albumin, immunoglobulin IgG, and II, VII, VIII, IX, X, XI, XIII coagulation factors and Van derrick's factor are in the S The growth factor thick solution obtained from the /D-treated initial platelet concentrate and the CaCl2-activated platelet concentrate was measured as t. These results are disclosed in Table 44 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) L15 <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 1344963 Table 3: Starting platelet concentrate (S/D-PC) obtained from 1% TnBP-1% Triton X-45 of the present invention, or starting platelet concentrate obtained by activation with CaCl2 (Act-PC) Comparison of various protein hanrosities in growth factor concentrates. Therefore, it is shown that the activated platelet concentrate almost completely removes fibrinogen and clotting factors. Π.3-bovine thrombin activation experiment

CaCl2/glass pellet treatment only partially activates platelets, which may explain the PDGF-AB'TGF-βΙ and EGF content in the release is lower than that in the S/D lysate; in order to break the above assumptions, The content of these platelet-derived growth factors in the release of platelet thick liquor 45 1344963 (third image), the release of the aforementioned platelet thick solution was first activated by CaCh, followed by bovine thrombin (Act_T) and coagulation Enzyme = further processing and further S/D treatment (Act_T_s/D). The second panel shows that PDGFAB (A), TGF-βΙ (B), and EGF (C) are activated in the initial platelet thickening (starting), after CaC12 activation (Act) followed by bovine thrombin activation ( Act-T) and S/D processing (Act_T_SD)

The amount of time. It can be seen that, in contrast to CaCU activation, bovine thrombin activation does not increase the release of pdgf ab (a) and TGf EGF (C). P Ting is the first to show that when S/D is treated with platelets, the release of platelet growth factor (especially the release of PDGF-AB'TGF-βΙ and EGF) will activate platelets with calcium and/or thrombin. 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 those platelet source growth may be lower, probably because agglutinated platelets and partially released, small plate source growth factors are caught in the fibrin network. In a platelet gel (called a platelet gel), the network is formed by the initiation of polymerization by fibrin in the thick liquid of the initial plate. Indeed, CaCi2 only, partial activation, and endogenous coagulase-induced blood clot formation of extraordinary collaterality, can be promoted in activated platelet release to add about 48 NIH units of bovine thrombin After /w, the fact that platelet-derived growth factor release continues to increase is still not ruled out. 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 when the amount of bovine thrombin was used, the amount of platelet growth factor in the effluent was Γ1 ̄ ̄ ̄ = now, 1GF_1 content in s/D After processing, it will be added. This thrombin activation only slightly increases the fact that most of the IGFs are free to circulate in the money, only a very small percentage of the 4 small plates are in our number*, and other methods such as thrombin activation, Cold Chen • I Eastern cycle and / or freeze-dried compared to S / D treatment 2, plate lysis is the most effective release of platelet growth factor from platelet cells to exudate means. In the early research, the average amount of PDGF in the whole blood serum was 17.5 ng/ml, that is, the amount of platelets containing 〇 〇 6 ng. This amount is equivalent to 6 ng/m of the separation platelet concentrate in our study and we found that the amount of pDGF in the S/D-treated slab thick solution was on average about three times higher. Our data further show that to increase the release of pDGF_AB, TGF-pj and EGF from platelets, 1% TnBp_1〇/〇 can be used.

Triton X-45 combination, 2% ΤηΒΡ, or i〇/0 triton X-45 or Triton X-100 S/D treatment, and oil extraction to remove S/D, does not reduce platelet dissolution The content of four platelet growth factors in the cell product. II. Comparison of lipid content in the growth factor thick solution After the initial platelet thick solution was activated by S/D or Caci2, the lipid content in the platelet growth factor thick solution was measured and compared with the starting platelet thick solution. The contents in the comparison are as shown in Table 4. 47 1344963 Cholesterol Triglyceride HHDL LDL (mg/dl) (mg/dl) (mg/dl) (mg/dl) Starting PC 164 170 33 99 Trit-PC 160 162 101 62 S/D-PC 1 32 20 11 0 S/D-PC 3 33 43 2 0 Act-PC 150 156 31 97 Table 4: Comparison of lipid content of growth factor concentrates prepared from the same starting platelet concentrate using 0.5% Triton (10) (Trit-PC); or after the S/D treatment of the present invention, followed by ~ poor oil extraction (S/D-PC 1); or after the S/D treatment of the present invention, three oil extractions (S /D-PC3); or after the platelet's caCl violates 2 /〇. Measuring blood lipid levels were tested by Hitachi clinical instrumentation. Each sample was based on pH 7.2 and contained 886 X lQ3 τ χ 1〇6 platelets/μΐ, 0.1 X 103 white blood cells (WBC) / μΐ and 〇〇 8 red blood cells (RBC) / pl. The initial platelet thick solution was prepared as shown in Table 4, and treated with Triton-treated platelets or prepared. In contrast to the platelets, in the S/D-PC growth factor concentrate prepared by S/D treatment, 'ldl (low a extract protein), HDL (still density lipoprotein), three Acid glycerides and phospholipids are significantly lower. Further, no significant difference was observed when one or three extractions were performed by S/D treatment of lysed blood. The plate is oil-extracted in the growth factor thick liquid of the present invention, and the cholesterol and the tri-acid LDL are removed, which will cause the clinical and therapeutic purposes to be reported as bribes and 48 J344963 because triglyceride and LDL-cholesterol combination are currently known. Atherosclerosis and the role of cardiovascular disease through the formation of fatty plaques in the arteries and venous systems that can cause heart attacks, strokes, and peripheral vascular disease. II.5-Growth factor activity Microscopic observation shows the change in shape, size and number of cells: when the cells are combined with platelet-derived growth factor concentrate obtained by S/D treatment or CaCh-activated initial platelet concentration solution When cultured, many spindle cells are specifically observed. Further, the cell count showed that, compared with Act-PC or cells not co-cultured with growth factor concentrate, when the cells were co-cultured with SD-PC growth factor concentrate, 'MG-63 osteoblasts The number will increase significantly in a time and dose dependent manner. MTT analysis also showed that the presence of SD-PC growth factor concentrate increased the cell viability of cultured cells compared to Act-PC treated cells. S/D-PC or Act-PC growth factor concentrates did not exhibit cytotoxicity to MG-63 osteoblasts. • The results of this study show that 'from 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 co-cultured with a growth factor concentrate obtained from S/D treatment, the cellular response is significantly increased. The effect of the concentrated solution obtained by the method of the present invention on MG-63 cells is improved, which may be due to an increase in the growth factor content, particularly PDGF, TGF-βΙ and EGF (these factors are in wound healing and tissue regeneration). The importance of the aspect is known to be 'and/or may be due to the increased amount of other biologically active substances that were not present in the activated jk platelet thick liquor or that exhibited a low amount of bioactive material. 49. The obtained fruit ί confirmed the important potential of the present invention or the method of the present invention for the use of the therapeutic substance and the preparation of the improved, Tianyue L medium. II.6-Virus Deactivation In addition, there is ample evidence that the treatment used here will =i deactivate the blood-derived envelope virus, especially HCV oxime, as indicated by the materials and methods above, 1% TnBP-l% Triton X-45 combined with 3PC treatment will ensure that the HIV, BVDV and PRV reduction factors are >5.6, >6.6, and > 6 4 within 5 minutes of treatment. In addition, the >7.0 loglQ vsv and Sindbis model virus will be quickly deactivated. Non-enveloped viruses (such as ΉΑν and small virus Β19) cannot be deactivated by §/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 nanotransition can also be used to reduce the risk of non-enveloped virus contamination. Obviously, the gold platelet growth factor formulation described herein will have to be produced under Good Manufacturing Practice (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). S/D treatment of such platelet formulations can provide a degree of practical advantage in topical applications. The safety of the first 'allogene-supplied virus 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 50 1344963 platelet release. 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 to use; or, in the case of allowing the use of recombinant platelet-derived growth factor (such as In order to treat some of the hypothalamic cancers, when it is combined with natural platelet-derived growth factors, a favorable 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 bovine serum, or in cell engineering. Research, or mesenchymal stem cells> lingual expansion (expansi〇n) and its differentiation into osteoblasts or chondrocytes are used as recombinant platelets & growth factors. ~ [Simple description of the diagram] The first picture: Study design: divide the blood of the donation into two subset pools. Subset pool 1 was treated directly with 1% TnBP-1% Triton X-45 for 1 hour without prior activation with CaCl:2. Sub-pool 2 was first activated with 23 mM CaCl2 and glass pellets to form a gold plate 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. Figure 2: (A) PDGF_AB, (B) TGF-βΙ, (〇EGF and (D) IGF in the initial platelet concentrate (start), in direct s/d treatment (1% TnBP_l% Triton X- 45) After (S/D), after initiation of platelet thick solution by CaCl2 activation (activation), and in CaCl2 activation 51 1344963 platelet release by S/D treatment (1% TnBP-1% Triton X- 45) Post-(activation + S/D) content (ng/ml). The s/D treatment is carried out as described in Materials and Methods and includes 3 oil extractions to remove the s/D agent. :(A) TOGF-AB, (B) TGF-βΙ and (C) EGF in the initial platelet concentrate (start), after activation of CaCb in the initial platelet concentrate (Act) followed by bovine thrombin activation (Act-T) and S/D treatment (1% TnBP-l% Triton X-45) (ActT-S/D) content (ng/ml). S/D treatment is carried out as described in Materials and Methods And includes 3 oil extractions to remove the S/D agent. Figure 4: Start gold plate thick liquid (line 1), the same platelet thick solution treated by the solvent-cleaner method of the present invention (2nd) Line) and the same platelet thick solution after CaCl2 activation (3rd) Comparison of protein compositions between rows. Samples were separated by SDS-PAGE on 4%-12% gels, after which the proteins were stained with Coomassie blue. Line M corresponds to protein labeling (size in thousands of Daltons) (kiloDalton), indicated on the left side of the gel.) Figure 5: Overdue frozen platelets are treated with S/D (1〇/〇)

TnBP-1% Triton X-45) (S/D-PC), or accepting CaCl2 activation (Act-PC), or selective activation in CaCl2 followed by S/D treatment (Act-PC+S/D) In the thick solution, the composition of PDGF_AB (figure 5 and EGF (figure b) is compared. The sixth circle: expired frozen platelets are treated with S/D (1%)

The compositions of igf_i (sixth figure a) and (sixth figure b) were compared in a thick liquid obtained by TnBP-1% Triton X_45) (S/D-PC) or by CaCi2 activation (Act-PC). [Main component symbol description] None 52

Claims (1)

April 6, 100, 100. Application for full-time®: 1. A virus-deactivated clotting platelet growth factor concentrate for therapeutic and/or cosmetic use. 2. For example, apply for a virus-deactivated clotting platelet growth factor thick solution containing the growth factor pDGF, TGM, IGF, EGF, CTGF, bFGF as described in item i of the full-time sub-paragraph i. A clotting platelet growth factor concentrate that is deactivated by the virus, characterized by the absence of a blood cell-associated transfusion reaction. 4. The virus-deactivated clotting platelet growth factor concentrate according to claim 1, further comprising at least one selected from the group consisting of fibroneetin, vitronectin, thrombin A protein consisting of a group of thromb〇sp〇ndin, v〇n Willebrand factor, and II, V, VII 'VIII, ιχ, χ, and χι coagulation factors. 5. A method of preparing a virus-deactivated clotable platelet growth factor concentrate comprising the steps of: a) contacting a starting platelet concentrate with a solvent and/or a cleaning agent; b) causing said starting platelet concentrate And co-cultivating the solvent and/or cleaning agent at a pH ranging from about 6.0 to about 9.0 and at a temperature ranging from 2 ° C to 50 ° C for at least 5 minutes to 6 hours; and The solvent and/or cleaning agent is removed 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 trialkyl phosphates having different alkyl chains. A group consisting of classes. 7. The method of claim 6, wherein the solvent is tri-n-butyl phosphate (ΤηΒΡ). 8. The method of claim 5, wherein the cleaning agent is selected from the group consisting of polyoxyethylene derivatives of fatty acids, partial esters of sorbitan, nonionic detergents, deoxygenated A group consisting of sodium cholate and sulfobetaines. 9. The method of claim 8, wherein the cleaning agent is Triton X-45, Triton X-100 or Tween 80. 10. The method of claim 5, wherein the respective final concentration of the solvent and/or the cleaning agent ranges from 0.2 to 5% by volume, which is the volume of the aforementioned starting platelet thick solution. Basic calculation. 11. The method according to claim 5, wherein the platelet thick solution is contacted with 2% ΤηΒΡ alone or with 1% ΤηΒΡ and 1% Triton Χ-45, which is the aforementioned initial platelet. The volume of the thick liquid is based on the basis. 12. The method of claim 5, wherein the oil extraction is carried out with a pharmaceutical grade oil, the amount of the oil being from 2 to 20% by weight, or from 5 to 15% by weight, or from 5 to 10 % by weight, based on the weight of the mixture of the aforementioned platelet concentrate and the aforementioned solvent and/or detergent. 13. The method of claim 5, wherein the chromatographic method comprises a C18 ceria loading material, or an SDR (Solvent-Detergent removal) hyper D. 】 民 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = The method described in claim 15 is to use the platelet separation h, ::: separation method from the whole blood force "乂 2 thick liquid system is fresh, expired" or expired and the blood is described The method of the method of Τ Τ Τ Τ Τ 液 液 用途 用途 去 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 == Peripheral growth factor thick liquid, or as claimed in the patent application; 可 之 可 用于 用于 用于 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗: The method described in Item 9 of the ninth item, which is from 〇 to I, the active dry circumference is from 2〇117/111丨 to 1〇〇〇%=: the clotting of the virus is activated. The method described in the 2nd item of the blood of the second section, the capping of the blood coagulation 2. The medical product, which is obtained by the method as described in the Patent Application No. 5 to 17 ^44963, Republic of China, April 6, 100 Disease Toxic = anti-growth factor thick solution, or as described in the scope of the patent application No. ^ ^, for therapeutic and / or cosmetic 23. Condensable platelet growth factor thick solution. Any of the range 5th to 17th. 24. The method of claim 5, wherein the method of applying the method of any one of the items 5 to 17 applies for a platelet-derived growth factor. For the use of viruses for deactivation of sexual use == 25. The use of vesicles for the regeneration of bone collaterals, or the healing of soft fibrils and hard tissues. - Examples of applications for patents 5 to 17 Any of the m sets of virus deactivated clotting platelet growth due to a thousand/thick thick liquid, or as used in any one of claims 1 to 4 for therapeutic and/or cosmetic materials The silk deactivated can be used for the thick liquid of the platelet growth factor, which is used for the cell culture of the living body 26 (bright η (10)) or the living body study WV 〇). Virus deactivated clotting platelet growth as described in any one of items 5 to 17 56