RU2743227C1 - A method of obtaining a complex of allogeneic lyophilized platelet growth factors for stimulating regenerative osteogenesis - Google Patents

Abstract

FIELD: medicine and pharmaceutics.

SUBSTANCE: invention relates namely to a method for producing a complex of allogeneic lyophilized platelet growth factors for stimulating regenerative osteogenesis, characterized in that it includes sampling whole blood, centrifuging blood at an acceleration of 350-750 g for 4-5 minutes at temperature 18-23°С, selection of the middle layer of plasma, repeated centrifugation of blood at an acceleration of 150-200 g for 3-4 minutes at a temperature of 18-23°С, selection of the supernatant layer, obtaining platelet concentrate, washing it with isotonic blood plasma solution, re-centrifuging it at an acceleration of 1000-2000 g for 4-5 minutes at a temperature of 18-23°C, taking a fraction of a "pure" platelet concentrate from the lower layer, freezing it by "shock freezing" with constant stirring for at least 15 minutes at a temperature of -30°C and below, then at a temperature of -80°C and below for at least 60 minutes, lyophilization and sterilization of the resulting complex.

EFFECT: increased concentration of growth factors without impurities.

1 cl, 1 tbl, 4 ex

Description

The invention relates to medicine, namely to traumatology and orthopedics, allows the production of a biologically active drug from alloblood to accelerate the processes of bone tissue regeneration in the body.

Known analogue: technology of lyophilization of platelet-rich plasma while maintaining the viability of factors TGF, PDGF, VEGF in the description of the invention to patent No. 2506946 RF, IPC A61K 9/19, A61K 38/18, A61L 15/44, B01J 3/00, according to application No. 2012151106/15 from 28.11.2012, publ. 02/20/2014, in bul. No. 25, including the collection of whole blood, its centrifugation, selection of the middle layer of plasma in such a way as to exclude the ingress of erythrocytes. The platelet concentrate obtained as a result of centrifugation is frozen in a refrigerator at a temperature lower than minus 1 ° C, and it is dried for at least three minutes in the temperature range from 2 ° C to 52 ° C; before use, the lyophilisate is sterilized.

Disadvantages: allows to obtain lyophilized platelet-rich plasma with insufficiently high concentration of growth factors and impurities.

The resulting lyophilized platelet-rich plasma can be used only in those patients from whose blood it was obtained due to the presence of immunogenic impurities, including antibodies and antigens, as well as cellular structures - leukocytes, since there is no washing stage during the manufacturing process. The presence of impurities in the finished product stimulates immune responses when administered to another person.

When using standard slow freezing, platelet growth factors in the concentrate are destroyed due to a gradual decrease in temperature and the transition of the substance from a liquid state to a solid. In addition, the lyophilization process takes place in a very wide temperature range from 2C ° to 52C °. At temperatures above 38 ° C, denaturation of protein subunits of growth factors is possible. Therefore, the final product has an insufficiently high concentration of growth factors. Since the sterilization of lyophilized platelet-rich plasma occurs immediately before use, and not immediately after preparation, during storage, foreign particles caught at the time of preparation, for example, during lyophilization, can worsen the properties of the resulting preparation.

Known analogue - a method of obtaining platelet-rich autoplasma in the description of the invention to patent No. 2305563 RF, IPC A61M 1/36, A61K 35/16, A61K 35/14, according to application No. 2005125929/15, from 15.08.2005; publ. 02/20/2007, in bul. No. 25, which consists in the introduction of a stabilizer into the autogenous blood, its centrifugation, the addition of a procoagulant containing a 10% solution of CaCl ₂ and a blood coagulation factor, characterized in that centrifugation is carried out at room temperature for 6-8 minutes at a speed of 1000-2300 rpm, and then 5000 U of prothrombin is added as a blood coagulation factor.

Disadvantages: allows you to get platelet-rich autoplasm (hereinafter referred to as autoBoTP) with impurities, with an insufficiently long shelf life.

The drug can only be used in a patient from whose whole blood it was prepared. When used in another patient, the likelihood of complications in the form of an immune reaction is high, since autogenous blood is used in the preparation of BOTP and the resulting preparation contains antibody protein units, antigens and cell structures such as leukocytes.

AutoPHP, which is in a liquid state, quickly loses its properties due to denaturation of protein structures and release of pro- and anti-inflammatory cytokines and other immunogenic structures that affect therapeutic efficacy and immune reactivity.

Known is the closest analogue - a method for producing an extract of allogeneic growth factors in the description of the invention to the patent EP Pat No. 13171495, IPC A61K 35/16; A61K 35/19; A61K 45/06; A61L 27/54; A61R 17/02, from 11.06.2013, in which the extract was obtained by separating platelets from plasma with their subsequent activation. The preparation of human plasma, consisting of platelets and plasma proteins, is carried out as follows: separating platelets from plasma and plasma proteins, mixing the platelets separated at the previous stage with distilled water to release growth factors, and separating the released growth factors to obtain an extract of allogeneic growth factors. Then, the isolated extract of allogeneic growth factors is lyophilized.

Disadvantages: allows to obtain an extract of allogeneic growth factors with impurities and insufficiently high concentration of growth factors.

There is no stage of obtaining platelet concentrate (hereinafter referred to as TC). At the stage of obtaining "pure" platelets, leukocytes remain in significant quantities during washing. And when platelets are activated to release growth factors from leukocytes, active substances are also released, which, upon repeated centrifugation, remain with platelet growth factors, since they have a similar molecular weight. This will induce local immune processes at the injection site.

The method provides for a stage at which platelets are washed and washed from plasma, followed by activation and further repeated repeated centrifugation and shaking, which stimulates the release of growth factors into the plasma, which is filtered out at the next stage, reducing the amount of growth factors in the preparation.

Before lyophilization of the obtained material, when using standard slow freezing, destruction or partial damage of platelet growth factors due to the crystallization stage is possible.

The drug can be used only in a patient from whose whole blood it was prepared due to the content of immunogenic impurities in the drug, which can cause an immune response of the body when used in another patient.

EFFECT: increased concentration of growth factors without impurities, with a long shelf life.

The technical result in a method for producing a complex of allogeneic lyophilized platelet growth factors for stimulating regenerative osteogenesis is achieved due to the fact that it includes sampling whole blood, centrifuging blood at an acceleration of 350-750 g for 4-5 minutes at a temperature of 18-23 ° C, selection of the middle layer of plasma, repeated centrifugation of blood at an acceleration of 150-200 g for 3-4 minutes. at a temperature of 18-23 ° C, selection of the supernatant layer, obtaining a platelet concentrate, washing it with a solution isotonic to blood plasma, re-centrifuging it at an acceleration of 1000-2000 g, for 4-5 minutes at a temperature of 18-23 ° C, sampling from the lower layer of the "pure" platelet concentrate fraction, freezing it by "shock freezing" with constant stirring for at least 15 minutes, at a temperature of at least -30 ° C, then at a temperature of at least -80 ° C, for at least 60 min., lyophilization and sterilization of the resulting complex.

In the claimed invention, a high concentration of platelet growth factors is achieved by a sparing attitude to cellular and protein structures. Initially, the obtained whole blood is centrifuged "gently" at an optimum acceleration of 350-750 g for 4-5 minutes at a temperature of 18-23 ° C, obtaining PRP. In contrast to analogues, the parameters used in centrifugation do not depend on the brand, model and configuration of the rotor and are defined as the acceleration of gravity g.

The resulting complex of allogeneic lyophilized platelet growth factors (hereinafter referred to as alloLTPR) to stimulate regenerative osteogenesis can be used in any patient by obtaining a "pure" drug. The "purity" of the complex is achieved by preparing TC immediately after receiving PRP immediately before washing. The stage of obtaining TC makes it possible to obtain the drug that is most pure from leukocytes. Since during centrifugation at an optimal acceleration of 150-200 g for 3-4 minutes at a temperature of 18-23 ° C, precipitation of leukocytes occurs, and the most viable and non-activated platelets remain in suspension in the supernatant. The resulting MC does not contain leukocytes, which at further stages of preparation can release antigens and antibodies into the concentrate, thereby reducing its immune resistance.

In the claimed invention there is a stage of washing the TC. The platelet concentrate is purified preferably with a saline solution of 0.9% NaCl or any other indifferent solution that does not affect blood components. This procedure allows platelets to be washed from unwanted particles, such as antigens, antibodies that remain in the plasma and can cause an immune response and an allergic reaction in a patient, including local immune processes in the field of drug administration.

In the claimed invention, the preparation is made from allogeneic blood, which is taken from a donor, which allows the preparation to be prepared in advance and used as needed in any patient.

In the claimed invention, in contrast to analogs, shock freezing of small volumes is used, in test tubes with a volume of 2-5 ml. Freezing takes place, for at least 15 minutes, at a temperature of at least -30 ° C, then at a temperature of at least -80 ° C, for at least 60 minutes.

This makes it possible to freeze alloLTPR in the shortest possible time without damaging their structures, since at slower freezing, damage to growth factors is possible.

In the claimed invention, a longer shelf life is assumed due to the use of lyophilization technology, which allows the preparation of a preparation without losing its structural integrity and biological activity. Lyophilization provides the preparation in a dried state, which, unlike analogs, is stored longer. With the proposed method of lyophilization, growth factors do not undergo denaturation and can persist for a long time. The alloLTPR obtained by adding an indifferent solution that does not affect blood components restore their original properties.

From the prior art, no technical solutions have been identified containing features that coincide with the distinctive features of the proposed method, therefore the proposed method meets the criterion of inventive step.

The presence of essential features distinguishing from the prototype allows the claimed technical solution to be recognized as new.

The possibility of implementing the claimed invention in industry allows us to recognize it as corresponding to the criterion of industrial applicability.

The claimed method is carried out as follows.

Due to the ability of lyophilized platelet growth factors to be stored for a long time, without the use of special storage conditions, the procurement of allogeneic lyophilized growth factors is carried out in advance.

Whole blood is taken from the donor in a volume of 200-500 ml with a platelet concentration of 180-320 × 10 ⁹ / l. Blood is collected in sterile tubes in compliance with the rules of asepsis, after adding the anticoagulant 3.8% sodium citrate. After filling the test tube with blood, it is closed with a sterile stopper and carefully, smoothly, several times inverted to mix the anticoagulant and blood. Then the blood is subjected to centrifugation at an acceleration of 350-750 g for 4-5 minutes. at a temperature of 18-23 ° C. Under sterile conditions, the middle layer of plasma is selected in such a way as to exclude the ingress of erythrocytes. The PRP obtained as a result of centrifugation is re-centrifuged at an acceleration of 150-200 g for 3-4 minutes. at a temperature of 18-23 ° C. Under sterile conditions, the supernatant is selected in such a way as to exclude the ingress of leukocytes from the bottom of the tube.

The MC obtained as a result of centrifugation is washed in an isotonic blood plasma solution, for example, saline solution of 0.9% NaCl or any other indifferent solution. 200-500 ml of 0.9% NaCl is added to the TC, then centrifuged again at an acceleration of 1000-2000 g for 4-5 minutes at a temperature of 18-23 ° C. Under sterile conditions, the lower layer of the fraction is sampled.

The resulting "pure" TC is distributed into test tubes, preferably with a volume of 2-5 ml, frozen for at least 15 minutes, at a temperature of at least -30 ° C, then at a temperature of at least -80 ° C, for at least 60 minutes The resulting material is lyophilized, sterilized using an ozone chamber. The obtained alloLTPR is transferred under aseptic conditions into an airtight container, stored in a dry place, without access to sunlight, until it is used. Lyophilization ensures that the preparation is obtained in a dried form, while maintaining all the properties that are available before lyophilization.

An example of the implementation of the proposed method.

Due to the ability of lyophilized platelet growth factors to be stored for a long time without special storage conditions, they are harvested in advance.

Whole blood is taken from the donor in a volume of 200-500 ml with a platelet concentration of 180-320 × 10 ⁹ / l. Blood is collected in sterile tubes in compliance with the rules of asepsis, after adding the anticoagulant 3.8% sodium citrate. After filling the test tube with blood, it is closed with a sterile stopper and carefully, smoothly, several times inverted to mix the anticoagulant and blood. Then the blood is subjected to centrifugation at an acceleration of 350-750 g, for an optimal time of 4-5 minutes at a temperature of 18-23 ° C. Under sterile conditions, the middle layer of plasma is selected in such a way as to exclude the ingress of erythrocytes.

At an acceleration of 350-750 g for 4-5 minutes, the maximum separation of whole blood into fractions is ensured. When taking the middle layer of plasma, the platelet content in it will be maximum, since most of the erythrocytes and leukocytes will be in the sediment. The worst results are obtained by centrifugation with an acceleration of less than 350 g, more than 750 g for less than 4 minutes, more than 5 minutes, at a temperature below 18 ° C, above 23 ° C. Since at an acceleration of less than 350 g for 4-5 minutes, the plasma layers are visually poorly determined and when the middle plasma layer is taken, a larger number of erythrocytes and leukocytes enter, which ultimately affects the "purity" and therapeutic efficacy of the obtained drug. When accelerating more than 750 g for 4-5 minutes, together with erythrocytes and leukocytes, a large number of platelets settle, as a result of which the middle plasma layer will be depleted in platelets, which ultimately will affect the concentration of growth factors in the drug, thereby reducing its effectiveness in application. When centrifuged with an acceleration of 350-750 g, less than 4 minutes. whole blood is not completely divided into fractions, which will lead to the selection of a large number of erythrocytes and leukocytes, thereby reducing the "purity" of the obtained preparation. When centrifuged with an acceleration of 350-750 g for more than 5 minutes, a large number of platelets settle, as a result of which the concentration of growth factors in the preparation decreases, thereby reducing its effectiveness when applied. A temperature from 18 ° C to 23 ° C helps to maintain platelet viability at all stages of preparation, a temperature decrease below 18 ° C and exceeding 23 ° C leads to the death or activation of platelets, which as a result minimizes the number of growth factors in the final preparation.

The PRP obtained as a result of centrifugation is re-centrifuged at an acceleration of 150-200 g for 3-4 minutes at a temperature of 18-23 ° C. Under sterile conditions, the supernatant is selected in such a way as to exclude the ingress of leukocytes from the bottom of the tube.

At an acceleration of 150-200 g for 3-4 minutes, the leukocytes remaining at the previous stage are precipitated, and most of the viable, not activated platelets remain in suspension. The worst results are obtained at acceleration less than 150 g, more than 200 g, for less than 3 minutes, more than 4 minutes, at temperatures below 18 ° C, above 23 ° C. Since at an acceleration of less than 150 g, more time is required for the sedimentation of leukocytes, and with an increase in time of more than 4 min, the release of pro-inflammatory cytokines into the plasma increases, which ultimately will affect the immunoresistant properties of the obtained preparation. At an acceleration of more than 200 g, together with leukocytes, precipitation of platelets also occurs, therefore, the resulting concentrate will contain a smaller number of platelets. When centrifuged with an acceleration of 150-200 g, less than 3 minutes. leukocytes do not completely precipitate and in the final MC there will be an increased number of leukocytes, thereby increasing the risk of obtaining unwanted particles in the form of antibodies, pro-inflammatory cytokines in the final preparation. A temperature from 18 ° C to 23 ° C helps to maintain platelet viability at all stages of preparation, a temperature decrease below 18 ° C and exceeding 23 ° C leads to the death or activation of platelets, which as a result minimizes the number of growth factors in the final preparation. The MC obtained as a result of centrifugation is washed. The MC obtained as a result of centrifugation is washed with a solution isotonic to blood plasma, for example, saline 0.9% NaCl or any other indifferent solution that does not affect blood components. In TC add 200-500 ml of 0.9% NaCl, then centrifuged again at an acceleration of 1000-2000 g for 4-5 minutes. at a temperature of 18-23 ° C. Then, under sterile conditions, the lower layer is selected.

When centrifuged at an acceleration of 1000-2000 g for 4-5 minutes, the platelets necessary to obtain the final substance precipitate, while smaller particles such as antibodies, pro- and anti-inflammatory cytokines and other immunogenic structures remain in suspension. The worst results are obtained by centrifugation with an acceleration of less than 1000 g, more than 2000 g, for less than 4 minutes, more than 5 minutes, at a temperature below 18 ° C, above 23 ° C. Since at an acceleration of less than 1000 g in 4-5 minutes, most of the platelets do not have time to settle, thereby the concentration of growth factors in the final product will be low. And at an acceleration of more than 2000 g in 4-5 minutes, damage and partial activation of platelets with the release of growth factors occurs, which ultimately will lead to their washing at subsequent stages. When centrifuged with an acceleration of 1000-2000 g for less than 4 minutes, most of the platelets do not precipitate and their concentration in the final product will be reduced. Centrifugation with an acceleration of 1000-2000 g for more than 5 minutes will lead to disruption of the platelet structure and their partial activation, which reduces the concentration of growth factors in the obtained preparation. A temperature from 18 ° C to 23 ° C helps to maintain platelet viability at all stages of preparation, a temperature decrease below 18 ° C and exceeding 23 ° C leads to the death or activation of platelets, which as a result minimizes the number of growth factors in the final preparation.

The resulting "pure" TC is distributed into test tubes, preferably with a volume of 2-5 ml, frozen by the method of "shock freezing": with constant stirring for at least 15 minutes, at a temperature of at least -30 ° C. For at least 15 minutes. at a temperature of at least -30 ° C with constant stirring, freezing occurs evenly in all areas. The worst results are obtained when freezing for less than 15 minutes, at a temperature above -30 ° C, since micro-areas of unfrozen material will remain, which ultimately contributes to the disruption of the lyophilization process and affects the quality of the resulting drug.

Then, immediately after the end of the first stage, the optimum temperature is reduced below -80 ° C for at least 60 minutes. If these conditions are met, the entire volume is completely frozen and the liquid substrate is easily sublimated, as a result, a higher-quality lyophilisate is obtained. The worst results are obtained at temperatures above -80 ° C, for less than 60 minutes, since the lyophilization process is disrupted due to uneven crystallization of the material.

The resulting material is lyophilized, sterilized using an ozone chamber. The obtained alloLTPR is transferred under aseptic conditions into an airtight container, stored in a dry place, without access to sunlight, until it is used.

In the claimed method, the obtained alloLTPR can be stored for a long time without losing its original properties, because lyophilization provides the preparation in a dried state, which, unlike analogs, is stored longer.

The method was tested in an experiment in two stages.

At the first stage of the experiment, blood was taken from laboratory animals, with further alloLTPR obtaining. The calculation of the required volume of whole blood for the preparation of LTFR was carried out taking into account that the PDGF content of 5-20 ng / ml is sufficient to stimulate the targeted action in regeneration. The volume of 0.5 ml of whole blood used for the preparation of LTFR is sufficient to have a positive effect on reparative osteogenesis.

In total, the experiment used 30 ml of whole blood, divided into 5 sterile tubes, 6 ml each, respectively, for each experimental subgroup.

The second stage of the experiment was carried out on 120 non-linear convection male rats 5-6 months old, weighing 450-550 g, divided into 10 subgroups.

1 group - experimental (60 rats), divided into 5 subgroups: 1 subgroup - assessment of osteogenesis on the 5th day (12 rats); Subgroup 2 - on day 14 (12 rats); 3 subgroup - for 21 knocks (12 rats); 4 subgroup - on day 32 (12 rats), subgroup 5 - on day 44 (12 rats).

Group 2 - control (60 rats) - 5 subgroups of 12 animals with an assessment of osteogenesis on days 5, 14, 21, 32 and 44, respectively.

Manual osteoclasia of the right femur was performed, forming a closed fracture with-n / 3 of the femur. AlloLTPR from each tube was diluted in 6 ml of 0.9% NaCl and the resulting solution was injected into the fracture area in a volume of 0.25 ml on the first and second days after osteoclasia. In the control group, a 0.9% NaCl solution in a volume of 0.25 ml was used similarly to the experimental group.

A comparative analysis of the treatment of bone fractures with the use of lyophilized platelet growth factors obtained from allogeneic blood and without the use of this drug by examining X-ray images, histological picture, and biochemical analysis was performed.

As a result, it has been proven that alloLTPR is able to stimulate reparative osteogenesis.

One of the main advantages of using alloLTFR is the possibility of long-term storage and preparation "in reserve". This will allow the drug to be used everywhere as needed.

AlloLTPR is not only able to normalize osteogenesis, as well as traditional auto-PRP, but can be used in patients weakened by somatic pathology and the severity of the condition, since only donor blood is used to prepare the drug.

The experiment carried out with the use of alloLTPR clinically, radiographically, morphologically, biochemically did not reveal any side effects. It can be stated that there is no reaction from the immune system in response to the administration of alloLTPR.

The carried out statistical processing of research results allows us to consider the effectiveness of using a complex of autogenous lyophilized platelet factors proven.

Examples of the use of a complex of allogeneic lyophilized platelet growth factors to stimulate regenerative osteogenesis obtained by the claimed method.

Example No. 1, the application of the claimed invention in the clinic, after clinical trials, may look like this.

Patient "R", 67 years old, trauma at home, falling from a step-ladder. Was admitted with a diagnosis of Closed comminuted fracture of both bones of the right leg in n \ 3 with displacement. History of closed compression fracture L _1-2 1 tbsp.

A patient in the trauma department underwent MOS of the tibia with a plate and screws. The fibula is fixed with needles. The synthesis is stable. Drainage. Hemostasis. Seams. The wound healed by primary intention, after 10 days the sutures were removed. The patient was discharged for outpatient treatment with recommendations.

After 8 weeks on the R-gr of the right shin, signs of pseudoarthrosis formation, repeated treatment.

5 weeks before the injury of patient "R" blood was taken from donor "M" in the amount of 300 ml. By centrifugation 400 g for 5 min at T-18 ° C. The resulting material was re-centrifuged at 170 g for 3 min at T 18 ° C. The resulting material was washed by adding 300 ml of 0.9% NaCl. Re-separation of platelets from other elements of the washed plasma by centrifugation of 2000 g for 4 min at T 18 ° C. Then the resulting material was distributed into 5 ml test tubes and frozen by “shock freezing with constant stirring for 20 minutes at a temperature of -30 ° C, then immediately after the end of the first stage, the temperature was lowered to -80 ° C, and frozen for 60 minutes. The resulting material was lyophilized, sterilized using an ozone chamber. Stored in a cool dry place.

AlloLTPR, previously diluted in 50 ml nat., Is injected into the area of forming pseudoarthrosis on the day of repeated treatment. solution on the first and second day. X-ray control after 6 weeks. Signs of the formation of periosteal callus.

Example 2.

Patient B, 52 years old, trauma in an accident. Was admitted with the diagnosis: Closed comminuted fracture of both bones of the left forearm in s \ 3 with displacement. A patient in the trauma department underwent open reduction, osteosynthesis of the left ulna and radius with titanium plates. The postoperative period was unremarkable. The wound healed by primary intention. She was discharged for outpatient treatment with recommendations. The patient independently removed the plaster cast 2 weeks after discharge. The load on the left limb was not dosed. I did not follow the recommendations of the attending physician. After 6 weeks on the R-gr of the left forearm, signs of delayed consolidation, repeated treatment. 3 weeks before the injury of patient "B" blood was taken from donor "O" in the amount of 350 ml. By centrifugation 450 g for 4 min at T-20 ° C. The resulting material was re-centrifuged at 180 g for 3 min at T 20 ° C. The resulting material was washed by adding 350 ml of 0.9% NaCl. Repeated separation of platelets from other elements of the washed plasma by centrifugation of 1500 g for 4 min at T 20 ° C. Next, the resulting material was distributed into 5 ml test tubes and frozen by “shock freezing with constant stirring for 15 minutes at a temperature of -35 ° C, then immediately after the end of the first stage the temperature was lowered to -90 ° C and frozen for 60 minutes. The resulting material was lyophilized, sterilized using an ozone chamber. Stored in a cool dry place.

In the area of delayed consolidation, the patient is injected on the day of re-treatment with alloLTPR, previously diluted in 50 ml of physical. solution on the first and second day. X-ray control after 5 weeks. Signs of the formation of periosteal callus.

Example 3.

Patient P, 57 years old, injury at work. Was admitted with a diagnosis of Closed fracture of 2, 3, 4, 5 metacarpal bones of the right hand with displacement. A history of type 2 diabetes mellitus for about 15 years. A patient in the trauma department underwent open reduction, osteosynthesis of 2, 3, 4, 5 metacarpal bones of the right hand with needles. The postoperative period proceeded against the background of a poorly healing postoperative wound. The stitches were removed for 14 days. Discharged for outpatient treatment with recommendations. After 4 weeks on the R-gr of the right hand, signs of delayed consolidation, repeated treatment. One week before the injury of patient "P", blood was taken from donor "C" in the amount of 300 ml. By centrifuging 500 g for 4 min at T-22 ° C. The resulting material was re-centrifuged at 200 g for 3 min at T 22 ° C. The resulting material was washed by adding 300 ml of 0.9% NaCl. Repeated separation of platelets from other elements of the washed plasma by centrifugation of 1500 g for 4 min at T 22 ° C. Then the resulting material was distributed into 3 ml test tubes and frozen by “shock freezing with constant stirring for 15 minutes at a temperature of -40 ° C, then immediately after the end of the first stage, the temperature was lowered to -90 ° C, and frozen for 60 minutes. The resulting material was lyophilized, sterilized using an ozone chamber. Stored in a cool dry place. In the area of delayed consolidation, the patient is injected with alloLTPR previously diluted in 20 ml nat. solution on the first and second day. X-ray control after 3 weeks. Signs of the formation of periosteal callus.

The use of the proposed method will provide a complex of allogeneic lyophilized platelet growth factors to stimulate regenerative osteogenesis with a high concentration of growth factors without impurities, a long shelf life.

Claims (1)

A method of obtaining a complex of allogeneic lyophilized platelet growth factors for stimulating regenerative osteogenesis, characterized in that it includes sampling whole blood, centrifuging blood at an acceleration of 350-750 g for 4-5 minutes at a temperature of 18-23 ° C, sampling the middle layer of plasma , repeated centrifugation of blood at an acceleration of 150-200 g for 3-4 minutes at a temperature of 18-23 ° C, selection of the supernatant layer, obtaining a platelet concentrate, washing it with a solution isotonic to blood plasma, re-centrifuging it at an acceleration of 1000-2000 g for 4-5 minutes at a temperature of 18-23 ° C, selection of a fraction of a "pure" platelet concentrate from the lower layer, freezing it by "shock freezing" with constant stirring for at least 15 minutes at a temperature of -30 ° C and below, then at a temperature of -80 ° C and below for at least 60 minutes, lyophilization and sterilization of the resulting complex.