RU2506946C1 - Technique of platelet-rich plasma lyophilisation with preserving tgf pdgf vegf factor viability - Google Patents

Abstract

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.

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Description

The invention relates to medicine, namely to traumatology and orthopedics, allows the manufacture of a biologically active drug from autologous blood to accelerate the regeneration of body tissues.

Known technology for the preparation and use of platelet-rich plasma (BTP) to stimulate the regeneration of living tissue (Robert E. Marx Platelet-reach plasma (PRP): what is PRP and what is not PRP? // Implant dentistry, Vol.10 No.4 2001). BOTP is obtained by centrifuging whole blood, followed by sampling a plasma layer over the separated red blood cells with a platelet concentration of at least 1 million / μl.

Growth factors are known - polypeptides with a molecular weight of 5-50 kDa, which have a wide range of biological effects. Growth factors, as a rule, are produced by non-specialized cells, including platelets. These include transforming growth factor (TGF), platelet growth factor (PDGF), vasculoendothelial growth factor (VEGF). TGF inhibits the synthesis of inflammatory cytokines, enhances the synthesis of proteins of the intercellular matrix, promotes wound healing, has anabolic effects, stimulates fibroblasts, the development of endothelium, is involved in the regulation of cell proliferation. PDGF is involved in the regulation of acute inflammation, wound healing and scar formation (found: http://laboratory.rusmedserv.com/files/39_Faktory_Rosta.pdf).

In the available literature, the question of the activity of these factors in living tissues or immediately after their isolation from cells and / or tissues is discussed, but no information was found about the preservation of activity after freezing and subsequent drying of blood serum.

A known preparation is a platelet concentrate of growth factors and a method for their preparation (WO / 2009/087560 dated July 16, 2009; PCT / IB2009 / 000013 GWO REI). The drug is intended for therapeutic and / or cosmetic use, contains growth factors PDGF, TGF-β, IGF, EGF, CTGF, bFGF and VEGF. A method of producing a platelet concentrate of growth factors in accordance with the invention includes the steps of contacting the platelet concentrate with a solvent and / or surfactants, incubating platelets with a solvent and / or surfactants for a period of at least 5 minutes to 6 hours. While the acidity (pH) is maintained in the range from about 6.0 to about 9.0, 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, it is necessary to use other active substances that are not desirable for the introduction into the patient's body, which can cause allergic reactions. There is no information on the possibility of long-term storage of the growth factors PDGF, TGF-β, IGF, EGF, CTGF, bFGF and VEGF obtained by this technology.

Known application JP 2011120763 "MATERIAL FOR REGENERATION TREATMENT" 06/23/2011; A61L 15/16; 2009281382. The development is aimed at obtaining materials for tissue regeneration using platelet-rich plasma and not requiring preparation before use or cryopreservation. The treatment regeneration material contains platelet-rich plasma, frozen and dried. The material is applied to the hard surfaces of instruments intended for osteosynthesis, used after storage in the refrigerator for at least one day, freezing and drying. The regeneration material can be used after the platelet-rich plasma has been 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, bulk materials, foam. To simulate continuous support structures, it is possible to produce adhesion receptors from biodegradable materials coated with a ligand of a cell substrate before applying a platelet-rich plasma.

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

EFFECT: development of lyophilization technology for platelet-rich plasma, which allows preserving the viability of such factors as TGF PDGF VEGF. A distinctive feature of the technology is the absence of exposure to chemical agents for the release of growth factors from platelets,

To implement the technique, whole blood is taken in an amount about three times larger than the volume of a defect in living tissue diagnosed by objective methods. In sterile tubes, following aseptic rules, they collect blood, after adding an anticoagulant, for example, sodium citrate, heparin or any other. After filling the tube with blood, it is closed with a sterile stopper and carefully, smoothly, turned over several times to mix the anticoagulant and blood. Then, the blood is centrifuged at a speed of 1000-1200 rpm for at least three minutes under hypothermia in the temperature range from 0C ° to 6C °. In sterile conditions, the selection of the middle layer of plasma is performed in such a way as to exclude the ingress of red blood cells. The platelet concentrate obtained as a result of centrifugation is a straw-yellow viscous liquid with a platelet content of about 800 thousand per microliter. Next, the concentrate is frozen in a refrigerator at a temperature lower than minus 1C °.

The frozen platelet concentrate is dried for at least three minutes in the temperature range from 2C ° to 52C °. The resulting lyophilisate is placed in an airtight container, stored in dry conditions. Before use, the lyophilisate is subjected to sterilization, for example, ozonation or irradiation with gamma rays.

Before direct use, the sterile lyophilizate is shaped in accordance with a tissue defect if bone tissue is to be replaced, or applied to a dressing for application to wounds on the body surface, or to metal structures intended for osteosynthesis or other supporting structures and materials for tissue regeneration . A possible variant of dilution in solutions intended for dissolving drugs for injection purposes, for example, in physiological saline sodium chloride, novocaine. The resulting solution is introduced into the zone with a deficiency of regenerative processes of bone tissue.

The method was tested using nine samples of donor blood taken from three practically healthy donors, three patients of the burn department with a thermal burn of at least 23% of the body surface; three patients in the intensive care unit with multiple injuries of the musculoskeletal system as a result of a traffic accident (accident). The results are presented in the table.

Table Protocol for assessing the content of growth factors in the lyophilisate of platelet-rich plasma Patient condition Growth Factor Content Tgf PDGF VEGF Almost healthy 1.386894 1,37987 1,934476 Almost healthy 2,694681 0.649351 2,418347 Almost healthy 1.451694 0,844156 1.952621 Burn 38% of the body surface 4.373597 3,766234 3.761089 Burn 33% of the body surface 2,229298 1,477273 2,977823 Burn 29% of the body surface 3,301447 2,621753 3,369456 Multiple Damage (Accident) 1,61075 0.381494 2,599798 Multiple Damage (Accident) 1,098238 0.698052 2,642137 Multiple Damage (Accident) 1.354494 0,539773 2,620968

The platelet-rich plasma lyophilizate prepared by our technology was used in the form of applications in dressings for the treatment of skin tissue defects and as injections at bone fracture sites from two weeks to one and a half months from the time of blood sampling. With each patient and his relatives, a preliminary discussion was held about the essence of the method, an agreement on informed consent was concluded. Wound healing occurred faster than provided by the target dates for such a volume and type of tissue damage.

Our technology for the preparation of platelet-derived growth factor lyophilisate allows us to obtain a new effect not known from the prior art: maintaining the viability of the growth factors TGF, PDGF, and VEGF obtained by their platelet-rich plasma for at least a month and a half from the moment of blood sampling from the patient.

Claims (1)

Lyophilization technology for platelet-rich plasma with viability of the TGF PDGF VEGF factors, including whole blood sampling, centrifugation, and selection of the middle plasma layer so as to prevent red blood cells from entering, characterized in that the platelet concentrate obtained by centrifugation is frozen in a refrigerator at a temperature below than minus 1 ° C, they are dried for at least three minutes in the temperature range from 2 ° C to 52 ° C; lyophilisate is sterilized before use.