Technique of platelet-rich plasma lyophilisation with preserving tgf pdgf vegf factor viability

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to traumatology and orthopaedics, enabling preparing a biologically active preparation of autoblood for enhancing neogenesis processes. The presented technique involves sampling whole blood, centrifuging, selecting a middle layer of plasma so that to avoid the erythrocyte ingress. The centrifuged platelet concentrate is frozen in a cold room at temperature below minus 1 C°, dried for at least three minutes within the temperature range of 2 C° to 52 C°; the lyophilisate is sterilised before use.

EFFECT: technique of platelet-rich plasma lyophilisation enables preserving the TGF PDGF VEGF factor viability min 1,5 months from the moment of blood sampling.

1 tbl

 

The invention relates to medicine, namely to traumatology and orthopedics, allows to produce biologically active preparation of autologous blood to accelerate tissue regeneration processes of the organism.

Known technology of preparation and use of platelet-rich plasma (PRP) stimulates regeneration of living tissues (Robert E. Marx Platelet-reach plasma (PRP): what is PRP and what is not PRP? // Implant dentistry, Vol.10 No.4 2001). PRP is obtained by centrifugation of whole blood with subsequent selection of a layer of plasma over useprofanity erythrocytes with a concentration of platelets at least 1 million/µl.

Known growth factors - polypeptides with molecular weight of 5-50 kDa, with a wide spectrum of biological actions. The growth factors, as a rule, are produced by specialized cells, including platelets. These include transforming growth factor (TGF), platelet-derived growth factor (PDGF), vascularendothelial growth factor (VEGF). TGF inhibits the synthesis of inflammatory cytokines, increases the synthesis of proteins of the extracellular matrix, promotes healing, and has anabolic effect and stimulates fibroblasts, the development of the endothelium, is involved in regulation of cell proliferation. PDGF is involved in regulation of acute inflammation, wound healing and scarring (found: http://laboratory.rusmedserv.com/files/39_Faktory_Rosta.pdf).

In the available literature, the discussion is about the activity of these factors in living tissues or immediately after separation from cells and/or tissues, but have not found information about saving activity after freezing and subsequent drying of serum.

A well-known drug, which is a concentrate of platelet growth factors and the method of their preparation (WO/2009/087560 from 16.07.2009; PCT/IB2009/000013 GWO REI). The product is intended for therapeutic and/or cosmetic use, contains the growth factors PDGF, TGF-β, IGF, EGF, CTGF, bFGF and VEGF. A method of obtaining a concentrate of platelet growth factors in accordance with the invention includes a stage of contacting the platelet concentrate with the solvent and/or surface-active substances, incubation of platelets with the solvent and/or surface-active substances for a period of not less than 5 minutes to 6 hours. Acidity (pH) is maintained in the range from about 6.0 to about 9.0 to, and at a temperature in the range from 2°C to 50°C, preferably in the range from 25°C to 45°C.

The disadvantages of the method: to obtain growth factors requires the use of other active substances that are not desirable for introduction into the patient's body, which can cause allergic reactions. There is no information about the possibility of long-term storage obtained by this technology is the AI of the growth factors PDGF, TGF-β, IGF, EGF, CTGF, bFGF and VEGF.

Known application JP 2011120763 MATERIAL FOR REGENERATION TREATMENT 23.06.2011; A61L 15/16; 2009281382. The development is aimed at obtaining materials for tissue regeneration using platelet-rich plasma and does not require preparation before use or cryopreservation. The material for the regeneration treatment contains platelet-rich plasma, frozen and dried. The material is applied on a solid surface tools for osteosynthesis, use after storage in the refrigerator at least one day, freezing and drying. Material for regeneration can be used after platelet-rich plasma was stored in the refrigerator for at least thirty days after freezing and drying. Tools for osteosynthesis can be selected from the group of textiles, porous materials, granular materials, foams. For structural modeling solid support can be made from biodegradable materials, coated with a ligand of a cellular receptor substrate adhesion prior to application of platelet-rich plasma.

The disadvantages of the method: no information about the degree of preservation of growth factors after freezing and drying of the platelet-rich plasma.

Technical result: development of technology lyophilizate the platelet-rich plasma, you can save the vitality factors such as PDGF TGF VEGF. A distinctive feature of this technology is the lack of exposure to chemical agents for the release of growth factors from platelets,

For the implementation of the methods produce intake of whole blood in an amount of about three times larger than diagnosed objective methods the volume of living tissue defect. In sterile tubes with observance of the rules of asepsis trying to enter the blood, after adding an anticoagulant, such as citrate, heparin, or any other. After filling the tubes with the blood of it is covered with a sterile tube and carefully, gently, overturn several times to mix the anticoagulant and blood. Next, the blood is subjected to centrifugation speed 1000-1200 rpm at least three minutes under conditions of hypothermia in the temperature range from 0C° to 6C°. Under sterile conditions make the selection of the middle layer of the plasma in such a way as to prevent the ingress of erythrocytes. Obtained by centrifugation of the platelet concentrate is a viscous liquid straw yellow color with platelets of about 800 thousand per microliter. Further, the concentrate is frozen in the refrigerator at a temperature lower than minus 1C°.

Frozen platelet concentrate is subjected to dry aniu for at least three minutes in the temperature range from 2C° to 52C°. Obtained by drying the freeze-dried is placed in an airtight container, store in dry conditions. Before applying the lyophilisate is subjected to sterilization, for example, ozone treatment or irradiation with gamma rays.

Before applying sterile lyophilisate shape corresponding to the tissue defect, if the substitution is subject to the bone, or put on a bandage for application to a wound on the body surface, or metal, designed for osteosynthesis or other auxiliary structures and materials intended for tissue regeneration. Possible breeding in solutions intended for dissolution of drugs with the purpose of injecting, for example, in a physiological solution of sodium chloride, procaine. The resulting solution is injected into an area with a shortage of the regenerative processes of bone tissue.

The method was tested using nine blood samples taken from three healthy donors, three patients in burn units with thermal burn not less than 23% of the body surface; three patients ICU with multiple injuries of the musculoskeletal system as a result of road traffic accidents (RTA). The results are presented in the table.

Table
Assessment Protocol the content of growth factors in freeze-dried platelet-rich plasma
The patient's conditionThe content of growth factors
TGFPDGFVEGF
Healthy1,3868941,379871,934476
Healthy2,6946810,6493512,418347
Healthy1,4516940,8441561,952621
Burn 38% of body surface4,3735973,7662343,761089
Burns 33% of body surface2,2292981,4772732,977823
Burns 29% of body surface3,3014472,6217533,369456
Multiple injuries (accidents)1,610750,3814942,599798
Multiple injuries (accidents)1,0982380,6980522,642137
Multiple injuries (accidents)1,3544940,5397732,620968

Cooked in our technology lyophilized platelet-rich plasma was used in the form of applications in the bandages for the treatment of defects of the skin tissue and injection in place of bone fractures in periods ranging from two weeks to six months from the date of blood sampling. With each patient and his relatives preliminary conversation about the essence of the method, the contract on informed consent. Wound healing occurred faster than the stipulated deadline for such amount and type of tissue damage.

The technology of preparation of freeze-dried platelet-derived growth factors allows to gain new, not known from the prior art effect: the preservation of the viability of the growth factors TGF, PDGF, VEGF from platelet-rich plasma for at least a month and a half since the blood of the patient.



 

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