UA113094C2 - METHOD OF OBTAINING AUTOLOGICAL FIBRIN GEL FOR STIMULATION OF BONE AND SOFT TISSUE REGENERATION AND REDUCTION OF INFLAMMATION - Google Patents

Abstract

The invention relates to orthopedics, traumatology and surgery and can be used to stimulate the regeneration of bone and soft tissues and reduce the intensity of inflammatory processes by using autologous fibrin gel when applied to the affected area. Autologous fibrin gel is obtained in a single-stage interaction of the blood plasma of a patient with the activator of prothrombin - the enzyme ecamulin, isolated from the venom of a multifaceted snake, in the presence of calcium ions.

Description

The invention belongs to medicine, namely to orthopedics, traumatology and surgery, and can be used to stimulate the regeneration of bone and soft tissues and reduce the intensity of the manifestation of inflammatory processes. Autologous fibrin gel allows to reduce allergy and immunogenicity, which increases the effectiveness of treatment. An autologous fibrin gel formed from the patient's blood plasma and the prothrombin activator - the enzyme ecamulin from the poison of the polychaete is injected into the contact area.

It is known about the use in clinical practice of compositions based on biopolymers (gelatin, collagen, fibrin, etc.) and polyesters (ethyl, n-butyl, 2-octyl, 2-cyanoacrylate), which have hemostatic and reparative properties. However, these composites have a number of disadvantages: a significant decrease in the ability to glue in the environment of the abdominal cavity, tissue irritation from certain types of adhesives that contain formaldehyde components (1-41.

It is known about the use of fibrin glue as a surgical adhesive or hemostatic agent in the form of special kits containing concentrated fibrinogen obtained from the blood plasma of donors, in combination with a bile activator of animal origin, such as thrombin or batroxobin, to obtain a heterologous fibrin glue (5-81 .

The main disadvantages of preparations made from blood plasma of donors are the possibility of developing allergic reactions to foreign proteins, the danger of transmission of hematogenous infections (hepatitis B, C and HIV infection), the complexity of production and use: multi-stage, multi-component, long-term preliminary preparation of the protein preparation, the possibility of spontaneous polymerization of fibrinogen in a vial.

The closest analogue of autologous fibrin gel is a fibrin sealant obtained from autologous blood |9Y, the production of which is carried out by obtaining fibrinogen concentrate from the patient's blood plasma or quarantined blood plasma of one donor. In the future, the necessary components (thrombin, calcium chloride, aprotinin) are added to the obtained concentrate and a fibrin sealant is obtained. The main disadvantages of this method are the use of large volumes of blood (60-150 ml), which limits the use of the sealant in children and patients with disorders of the hemostasis system, the complexity of manufacturing (multistage), the possibility of transmission of hematogenous viral infections with the thrombin preparation (hepatitis B, C and

HIV infection), the difficulty of determining the output of fibrinogen, which is associated with the preservation of conditions

From sterility. In addition, a special device - a two-section syringe - is required to apply fibrin sealant to the contact area.

It is known that bone regeneration during disease and bone fractures occurs more efficiently in the presence of biologically active low molecular weight protein components of blood plasma |101I.

The object of the claimed invention is: to create an easy-to-implement, one-step method of obtaining autologous virus-safe fibrin gel (hereinafter - fibrin gel) for clinical use immediately before use in orthopedics, traumatology and surgery using a kit for obtaining fibrin gel.

The problem of the claimed invention is solved by the interaction of the patient's blood plasma with the enzyme ecamulin as a (prothrombin activator) in the presence of calcium ions, which ensures the formation of thrombin in the blood plasma and, in turn, leads to the conversion of fibrinogen to fibrin with the formation of a fibrin gel, which has adhesive properties; and including the necessary sterile components in the kit for the preparation of fibrin gel: lyophilized enzyme ecamulin, calcium chloride solution and physiological saline.

Fibrin gel from the patient's own blood plasma helps stop bleeding, regenerate bone and soft tissues, reduces the intensity of inflammatory processes, eliminates the risk of infection, rejection and the threat of allergic reactions, ensures non-antigenicity and the absence of an irritating effect, is characterized by a high degree of modeling of the affected tissue surface and high density of adhesion to them, ease of application. Performing the method of obtaining fibrin gel requires a small amount of the patient's blood - 5-20 ml; the rate of fibrin gel formation depends on the amount of the enzyme ecamulin (0.10-1.00 mg) added to the blood plasma, the time of its formation lasts from 50 to 100 seconds.

Ekamulin is an enzyme that is secreted from the venom of the polychaete snake E. tiyizdoatatsiv (11.

The enzyme ekamulin is a lyophilized powdery substance of white color, easily soluble in water, and is an activator of prothrombin.

To prepare an autologous composition of fibrin gel, plasma is first obtained from the patient's blood, to which a solution of the enzyme ecamulin and a solution of calcium chloride are added; the mixture is carefully mixed and applied directly to the affected surface.

The kit for obtaining fibrin gel includes sterile components: lyophilized bo enzyme ekamulin; calcium chloride solution; physiological solution.

Example 1. The method of obtaining fibrin gel.

To obtain autologous blood plasma, immediately before the application of fibrin gel, the patient's blood is collected from the vein with a vacutainer in the amount of 5-20 ml, depending on the size of the affected area; blood is centrifuged on a general-purpose preparative centrifuge for 10 minutes. with an acceleration of 1200-1400 9, then the blood plasma, which is in the supernatant, is transferred to a plastic tube.

To obtain a fibrin gel, 0.1 ml of a solution of the enzyme ecamulin in a physiological solution with a concentration of 0.15 mg/ml and 0.1 ml of a calcium chloride solution with a concentration of 5 96. After 6025 s from the moment of adding the enzyme ecamulin, the mixture is transferred to the affected area, distributing it evenly, where the time of formation of fibrin gel from the moment of mixing the components is (70210) s. If necessary, the time of formation of fibrin gel can be changed by using a different amount of enzyme ecamulin

The yield is quantitative: 1.2 ml of fibrin gel is obtained from 1.0 ml of blood plasma.

Example 2.

Preparation of fibrin gel is carried out as described in Example 1, but at the same time, 0.1 ml of a solution of the enzyme ekamulin in physiological solution with a concentration of 1.0 mg/ml is added. After 4055 s from the moment of adding the enzyme ekamulin, the mixture is transferred to the affected area, distributing it evenly, where the time of formation of fibrin gel at such a concentration of ekamulin from the moment of mixing the components is (505) s.

The yield is quantitative: 1.2 ml of fibrin gel is obtained from 1.0 ml of blood plasma.

Example 3.

The production of fibrin gel is carried out as described in Example 1, but at the same time, a different amount of the enzyme ecamulin is added, namely: 0.1 ml of a solution of the enzyme ecamulin in physiological solution with a concentration of 0.10 mg/ml. After 80-410 s from the moment of adding the enzyme Ekamulin, the mixture is transferred to the affected area, distributing it evenly, where the time of formation of a fibrin gel at such a concentration of the enzyme Ekamulin from the moment of mixing the components is (90-10) s. The yield is quantitative: 1.2 ml of fibrin gel is obtained from 1.0 ml of blood plasma.

Example 4. Stimulation of reparative osteogenesis and reduction of inflammatory intensity

From processes when using fibrin gel.

Approbation of the claimed method is carried out on rabbits aged 7 months and weighing 2 kg.

Experiments are carried out on a model of a fracture of the radius bone by carrying out an osteotomy of the diaphysis of the radius bone.

All experimental animals after modeling the fracture are divided into two groups: experimental and control. The animals of the research group, after carrying out osteosynthesis, before applying sutures to soft tissues in the fracture zone, apply a composition for obtaining fibrin gel 40-60 seconds after mixing the components. When applying fibrin gel to the fracture site, its high adhesiveness and density of adhesion to the affected surface are noted. Animals of the control group were left without applying fibrin gel to the fracture area.

According to clinical and X-ray data, on the 3rd day after osteotomy, all animals showed pronounced signs of inflammatory reaction of surgical wounds, in particular, swelling, redness, and tenderness of the injured tissues. Animals do not rest on injured limbs.

Transverse fractures with a well-defined fracture line are determined X-ray (Fig. 1, 2). No lysis of the edges of bone fragments is observed. There are no reactions from the endo- and periosteum.

On the 18th day after the osteotomy, the animals of the experimental group were noted to have no signs of inflammation, a moderate periosteal reaction of the fracture zone, the formation of a connective tissue callus with well-defined processes of osteosclerosis (mineralization) (Fig. 3), and the animals were fully resting on the limb were observed radiologically.

On the other hand, the animals of the control group for this period after the osteotomy noted pronounced swelling of the soft tissues (Fig. 4) and their moderate tenderness during palpation. Radiologically, a massive, uneven periosteal reaction localized outside the defect zone is noted.

Weakly expressed tissue-specific structures are observed in the area of the defect with the initial stages of their mineralization.

When using fibrin gel on affected tissues of a living organism, its non-antigenicity, lack of irritating effect on adjacent tissues, high degree of modeling of the affected surface, ease of application, and rapid formation of a water- and air-tight jelly-like gel are noted.

The intensive reduction of the inflammatory process at the site of application of the autolytic fibrin gel, compared to the control, indicates the absence of the claimed irritating and immunostimulating effect of the gel.

Thus, it is shown that the application of the claimed fibrin gel stimulates the reparative processes of osteogenesis and helps reduce the inflammatory process.

Example 5. Stimulation of liver tissue regeneration.

The operation is performed by laparotomy on purebred dogs. An incision 4 cm long and 0.8-1 cm deep is made on the lateral side of the right lobe of the liver. In control, after the bleeding has stopped, catgut sutures are applied (Fig. 5). In the experiment, the composition for obtaining fibrin gel is applied to the liver wound 30-40 seconds after mixing the components (Fig. b).

The animals are observed and a relaparotomy is performed on the 6th day to analyze the condition of the sutures. The main distinguishing feature of a dog's liver wound in an experiment on the 6th day after surgery is the adhesion of the omentum to the surface of the wound in the case of using fibrin gel (Fig. 7).

The omentum fuses with the liver capsule in a thin strip only in the incision area and is easily dissected, leaving minor erosive defects of the liver capsule and capillary bleeding that stops on its own. Visually, the parenchyma of the liver in the zone of the surface of the wound does not differ significantly from the healthy one. In this area, a typical organic pattern is already visible on the surface, and it itself acquires a dark red color. A thin (about 1 mm) light strip is visible only along the cut line under the capsule itself, which indicates a moderate development of connective tissue. Seams are poorly visualized, they can be seen only by "impressions" on the liver capsule.

In the control, adhesive phenomena from the peritoneum or omentum of the dog are not observed (Fig. 8).

However, a more powerful connective tissue reaction is noted, which is pronounced on the side of the liver capsule. So, the latter is significantly thickened not only along the incision site, but also goes beyond the boundaries of the applied seams and completely covers them from above, in connection with which it protrudes above the surface in the places of tied knots. Along with this, the liver parenchyma around the injured area has a lighter color, which indicates a significant development of the postoperative inflammatory process and associated degenerative-dystrophic changes in hepatocytes. Organic drawing in

It is not visible from this area. Along with this, the seams themselves, which are currently only at the initial stage of their resorption, are well visualized. At the same time, a powerful connective tissue reaction is noted around them.

Thus, the macromorphological characteristics of liver wound healing indicate that the use of fibrin gel contributes to the rapid healing of a liver wound, mainly due to the activation of parenchymal regenerative potential and at least connective tissue potential. At the same time, fibrin gel limits and moderately reduces the development of a post-traumatic inflammatory reaction, which visually indicates a much smaller area of dystrophic lesions.

Example 4. A set of ingredients for obtaining the claimed fibrin gel.

When performing the claimed method, a kit is used for obtaining fibrin gel from 10 ml of blood plasma, which includes the following sterile components: - lyophilized enzyme ekamulin in vials -0.10; 0.15; 1.00 mg; - physiological solution in an ampoule - 1.0 ml; From the ampoule; - calcium chloride solution of concentration 5 95 in ampoule - 1.0 ml; From the ampoule.

Add 0.1 ml of physiological solution to a bottle with freeze-dried enzyme ekamulin; the reagent is ready for use in 20 minutes. after dissolution.

Thus, a new original one-stage method of obtaining autologous fibrin gel was created to stimulate the regeneration of bone and soft tissues and reduce the intensity of inflammatory processes in humans and animals using a kit for obtaining autologous fibrin gel.

The advantages of the claimed method are: - one-stage preparation of fibrin gel from the patient's blood plasma immediately before clinical use, which greatly simplifies the technology and shortens the time of its preparation compared to analogues; - a small sample of the patient's blood - 10-20 ml (against 60-150 ml, as in the closest analogue). - rapid formation of water- and air-tight autologous fibrin gel on the affected surface (45-100 s from the moment of mixing the components); - infectious safety due to the use of the enzyme ecamulin as an activator of 60 prothrombin (instead of thrombin, as in analogues);

- non-antigenicity and lack of irritating effect of autologous fibrin gel on adjacent tissues; - ease of application, high degree of modeling of the affected surface, high density of fibrin gel adhesion to tissues; - stimulating the regeneration of bone and soft tissues and reducing the intensity of inflammatory processes in humans and animals; - use of a kit for obtaining autologous fibrin gel.

Autologous fibrin gel can be recommended for use in orthopedics, traumatology, and surgery.

Sources of information: 1. Istranov L.P., Aboyants R.K., Istranova E.V. Local hemostatic agents based on collagen Surgery - 2006. - T. 08, Mo. 2. - P. 4-8. Author's certificate of the Russian Federation Mo 725289 dated July 31, 1975; patent of the Russian Federation No. 2156140 dated November 17, 1999 2. Popov V.A., Syrotinkin N.V., Holovachenko V.A. Latex tissue glue and its application in surgery. - Polymer and medicine. - 2006. - 1. - b. 25-26. 3. VO 2157243, publ. 10.10.2000. 4. VO 2242987, publ. 27.12.2004. 5. Paradise. 05 8491564, dshu 23, 2013. 6. Arr. 05 20130299407 JSC, Mom. 14, 2013. 7. Pat. YuA 10419, publ. 25.12.1996. 8. VO 2193868, publ. 10.12.2002 9. Pat. VO 2143924, publ. 10.01.2000. 10. O.L. Grebneva, D.V. Samusenko, M.A. Kovynka, S.N. Luneva Component! Fractionated blood plasma and their role in the mechanism of optimization of reparative osteogenesis // Geny of Orthopedics. - 2013. - Mo 2. - b. 102-105. 11. Soloviev D.A., Platonova T.N., Ugarova T.P. Isolation and characterization of zkamulin - a prothrombin activator from the venom of the multi-scaly Espiv tyizdataiiyiv5. // Biochemistry. - 1996. - T.61. - vpp. 6. - pp. 1094-1105.

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Claims (2)

FORMULA OF THE INVENTION 1. The method of obtaining autologous fibrin gel by the interaction of blood plasma with an enzyme in the presence of calcium ions, which differs in that the autologous blood plasma interacts with the enzyme ecamulin. 2. The method according to claim 1, which differs in that the autologous blood plasma of a person or an animal is mixed with a solution of the enzyme ecamulin with a concentration of 0.10-1.00 mg/ml at a volume ratio of 1:0.1, adding 0.1 ml of a calcium chloride solution with a concentration of 5.95. C. The method according to claim 1, which differs in that a kit is used for obtaining autologous fibrin gel, which contains sterile components: - lyophilized enzyme ekamulin in vials, mg -0.01;:50.15; 1.00; - calcium chloride solution with a concentration of 5 9o in ampoules, ml - 1.0; - physiological solution in ampoules, ml - 1.0.