RU2527701C1 - Method for preparing agent possessing property of cartilaginous, osseous, muscular tissue regeneration stimulation, and method for cartilaginous, osseous, muscular tissue regeneration stimulation with using prepared agent - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: what is described is a method for the cartilaginous, osseous, muscular tissue regeneration stimulation involving the setting of a long bone fracture and introduction of the prepared agent into the intramedullary canal and the surrounding muscular tissue; that is combined with the tissue regeneration stimulation by introducing a mixture of aqueous solutions of recombinant beta interferon (IFN-β) in the concentration of 1∙10⁵ units/ml and polyethylene glycol with a molecular weight of 6,000 (PEG 6000) in the concentration of 0.03∙10^-9 mole/ml in the relation of 1:4 respectively; that is followed by a retrograde intramedullary osteosynthesis of the long bone fracture.

EFFECT: agent enables providing higher clinical effectiveness in the long bone fractures, reducing injuries, simplifying preparation technology of the agent for the tissue regeneration stimulation within the fracture, and reducing a rate of pyoinflammatory complications.

10 dwg

Description

The alleged invention relates to medicine, namely to the creation of a method with properties that stimulate the regeneration of cartilage, bone, muscle tissue in the treatment of fractures of long bones of the extremities.

During ontogenesis, growth factors are expressed that can trigger the fundamental processes of tissue differentiation and growth. However, in a growing and mature organism, the ability to regenerate structurally and functionally damaged tissues is largely lost. The decrease in the ability to regenerate is due to a decrease in the expression of growth factors, which, in turn, control the expression of proteins necessary for tissue synthesis. Among the numerous endogenous factors with control and regulatory functions, an important place is given to interferons (IFN).

Interferons belong to the family of proteins that have various effects on target cells with antiviral, immunomodulatory activity. The following subtypes are distinguished depending on the producer cells: alpha subtype, beta subtype, gamma subtype (1).

Interferon beta has an effect on various cells of the body, being a mediator of the immune response, including the expression of molecules of the main histocompatibility complex, cytokine receptors for tumor necrosis factor, interleukins, colony stimulating factors and other growth factors (2).

Known means of a wide spectrum of action, used including to stimulate the regeneration of cartilage and connective tissue. A preparation containing human recombinant alpha, and / or beta, and / or gamma types interferon and an antioxidant complex of tocopherol acetate and ascorbic acid or its salts further comprises chondroitin sulfate, glucosamine. The polymers used, for example, are polyvinyl alcohol, polyethylene oxides having the properties of stimulating the synthesis of connective, cartilage, and bone tissues (RF patent No. 2349339, IPC A61K 38/21, A61P 37/02, A61P 19/02, published March 20, 2009 , Bull. No. 8), (3).

However, this drug is multicomponent with a wide spectrum of action, made in the form of a gel, ointment, cream, film and is used in the complex treatment of diseases of an infectious and inflammatory nature, accompanied by destructive processes or a predisposition to them.

Known tool polyethylene glycol (PEG) - a chemical substance that has properties that allow you to use it as a carrier for bioorganic molecules, such as proteins, and create a depot of the drug substance. Therefore, the introduction of drug compounds with PEG helps to improve the pharmacokinetic and pharmacodynamic properties, increase the half-life and, accordingly, reduce the fluctuations in the concentration of the drug in the body, increase its stability and biological activity (patent RU 2 433 135), (4).

There are various methods of stimulating the regeneration of structurally and functionally damaged tissues using various drugs and the technology of their application.

So, there is a method of stimulating reparative osteogenesis in bone fractures. The method consists in injecting soft tissues and / or subcutaneously in the projection of the pathological focus of the dispersed alloplant biomaterial, diluted in physiological solution in a ratio of 50 mg of biomaterial per 5-15 ml of solution, in an amount of 1-10 injections into the adjacent to the site of fracture or false joint. for one session of 0.1-15 ml per injection, the number of sessions from 1 to 10 with an interval of 1-7 days (RF patent No. 2364361, IPC АВВ 17/56, publ. 20.08.2009, Bull. No. 23), (5).

The disadvantages of this method include the fact that the number of injections can be from one to ten, which is determined by the characteristics of the fracture. In addition, the treatment is carried out in courses with the number of sessions 1-10 per course with an interval between sessions of 1-7 days, which significantly increases the duration of treatment.

Also known is a method of stimulating reparative osteogenesis in the replacement of bone defects resulting from trauma or after bone resection. The method includes injection implantation into the bone damage zone of cultured autologous bone marrow stromal progenitor cells. In this case, the implantation of cells is carried out at the end of inflammation in the area of bone damage and the onset of natural osteogenesis, 3-5 times with an interval of 2-3 days (RF patent No. 2373883, IPC АВВ 17/56, published on November 27, 2009 Bull. No. 33 ), (6).

The disadvantages of this method are:

- an operative or puncture path for taking part of the bone marrow in patients, which is associated with additional trauma;

- the complexity of the technology for the preparation of suspensions of autologous bone marrow stromal progenitor cells, which requires additional costs due to the need to use appropriate equipment and expensive reagents.

Closest to the proposed one is a method for optimizing reparative osteogenesis, including introducing into the bone marrow canal a fracture site or zone of the false joint of the pre-ground alloplant biomaterial for scleroplasty with autologous blood in the form of a suspension of 5 crushed alloplant biomaterial plates in 5 ml of autologous blood. The suspension is administered once by injection with a Gordeev needle with a hole diameter of 2 mm (RF patent No. 2315580, IPC A61B 17/56, publ. January 27, 2008 Bull. No. 3), (7).

The authors studied the possibility of optimizing the reparative regeneration of bone tissue under the conditions of an experimental fracture of long tubular bones with instability of fragments and the presence of diastasis of more than 0.5 mm between them by the combined use of pre-ground alloplant biomaterial for scleroplasty and autologous blood.

The disadvantages of this method are:

- the possibility of aseptic disturbance associated with the need for preliminary grinding of the biomaterial plates into crumbs to obtain a suspension;

- limiting the use of this method in patients with chronic infections (for example, viral hepatitis B, C, etc.) due to the inability to procure and use autologous blood;

- trauma due to the large size of the Gordeev needle and the need for access to the bone marrow canal.

The above was the basis for the development of a new method of treatment based on interferon-beta and polyethylene glycol (PEG 6000), which has additional properties necessary to stimulate the regeneration of cartilage, bone, muscle tissue with damage to the long bones of the extremities.

Based on the existing level of technologies for stimulating tissue regeneration during fractures, as well as the means used, and eliminating the disadvantages of known methods, the task was set: to increase the effectiveness of treatment for fractures of long bones, reduce its invasiveness by selecting the optimal concentration of the drugs used, simplify the technology of stimulating tissue regeneration in fracture zone, as well as reduce the frequency of purulent-inflammatory complications. The problem is solved as follows.

A method of stimulating the regeneration of cartilage, bone, and muscle tissue involves the reposition of a fracture of a long bone and the introduction of the prepared product into the bone marrow canal and surrounding muscle tissue. New in the proposed method is that they stimulate tissue regeneration by introducing into the bone marrow channel and surrounding muscle tissue a mixture of solutions of recombinant interferon beta (IFN-β) at a concentration of 1 ∙ 10 ⁵ IU / ml and polyethylene glycol with a molecular weight of 6000 (PEG 6000 ) with a concentration of 0.03 ∙ 10 ^-9 mol / ml in a ratio of 1: 4, respectively, after which retrograde intramedullary osteosynthesis of a long bone fracture is performed.

We explain the essential distinguishing features of the proposed method having properties that stimulate the regeneration of cartilage, bone, muscle tissue, and a treatment method using the prepared means.

It is the use of the agent by mixing aqueous solutions of recombinant interferon beta (IFN-β) at a concentration of 1 ∙ 10 ⁵ U / ml and polyethylene glycol with a molecular weight of 6000 (PEG 6000) as a polymer carrier with a concentration of 0.03 ∙ 10 ^-9 mol / ml in a ratio of solutions of 1: 4, respectively, allows you to create a depot of a medicinal substance and prolong its action, providing a long biological effect with a single use.

Traditional methods of administration of interferon (IF) preparations are characterized by rapid absorption from tissues, a large volume of distribution, low stability, a short half-life, which limits the effectiveness of their action. In this connection, in order to maintain therapeutic concentrations, there is a need for frequent administration of the drug in high doses, which can lead to undesirable side effects.

The use of polyethylene glycol with a molecular weight of 6000 (PEG 60,000) as a polymer carrier at a concentration of 0.03 × 10 ^-9 mol / ml ensures the prolongation of the biological effects of interferon-beta.

Performing stimulation of the regeneration of cartilage, bone, muscle tissue by a single injection of a mixture of aqueous solutions of recombinant interferon beta (IFN-β) and polyethylene glycol in a volume of 0.1 ml containing 2000 IU of interferon beta (2 ∙ 10 ³ IFN-β) provides long-term effects on the reparative process in the area of bone trauma with stimulation of bone and cartilage tissue growth and stimulation of muscle tissue regeneration.

The implementation of retrograde intramedullary osteosynthesis of a long bone fracture after a single injection of the above prepared means improves microcirculation in the fracture zone by stimulating angiogenesis.

Patent studies on subclasses A61K 38/21, A61P 19/00, A61B 17/56 and analysis of scientific and medical information reflecting the current level of methods for stimulating the regeneration of cartilage, bone, muscle, tissues, and the treatment method using the prepared preparation did not reveal identical technology. Thus, the proposed method and its use for the treatment of fractures of long bones is new.

The interconnection and interaction of the essential techniques of the proposed method, which has the property of stimulating the regeneration of cartilage, bone, muscle tissue, and the treatment method using the prepared means provide a new result in solving the problem, namely, to increase the effectiveness of the treatment of fractures of long bones, reduce its invasiveness by selecting optimal concentration of the drugs used, simplify the technology of stimulating tissue regeneration in the fracture zone.

The technical result is ensured by the fact that the agent having interferon-beta activity and a prolonged action due to the use of polyethylene glycol helps to improve microcirculation around damaged tissues in the fracture zone due to the stimulation of angiogenesis. The implementation of retrograde intramedullary osteosynthesis of a long bone fracture after a single injection of the proposed agent stimulates structural regeneration of cartilage, bone, muscle tissue.

A positive achievement of the proposed method is:

- decrease in the frequency of purulent-inflammatory complications;

- accelerated formation of structural bone callus in the fracture area;

- increased enchondral ossification;

- stimulation of angiogenesis;

- stimulation of myogenesis.

The proposed method using an agent having IFN-β activity and a prolonged effect due to the use of polyethylene glycol allows to obtain a positive effect - a long-term effect on the reparative process in the area of bone injury with stimulation of bone and cartilage tissue growth and stimulation of muscle tissue regeneration with a single application of the active substance .

Thus, the proposed method for stimulating the regeneration of cartilage, bone, and muscle tissue using an agent prepared from recombinant IFN-β and a high molecular weight polymer, polyethylene glycol with a molecular weight of 6000 (PEG 6000), has an inventive step.

The proposed method of stimulating the regeneration of cartilage, bone, muscle tissue using the prepared product is promising and can be widely used in clinical practice, in the departments of traumatology and orthopedics.

The essence of the proposed intellectual property is illustrated by illustrations, which show:

figure 1 - morphological photograph of the fracture zone in the animal of the control group on day 9, visible fibrous callus (position 1),

figure 2 is a morphological photograph of the fracture zone in the animal of the experimental group on day 9, the corns represented by fibrous (position 1), cartilage (position 2) and periosteal callus (position 3) are visible

figure 3 - morphological photograph of the fracture zone in the animal of the control group on day 14, visible fibrous callus (position 1) with zones of cartilage tissue (position 2),

figure 4 is a morphological photograph of the fracture zone in the animal of the experimental group on day 14, a small zone of fibrous callus (position 1), an extensive zone of cartilage tissue (position 2), a large area of periosteal callus (position 3) are visible,

5 is a morphological photograph of the fracture zone in the animal of the control group on the 35th day, the remains of the cartilage corns (position 2), the formed bone beams (position 4) are visible,

6 is a morphological photograph of the fracture zone in the animal of the experimental group on the 35th day, the formed bone beams are visible (position 4),

Fig.7 is a morphological photograph of the fracture zone in the animal of the control group on day 9, where CD34 + cells in the regenerate zone were detected (position 5), immunohistochemical staining for CD34 +, 400 magnification,

Fig. 8 is a morphological photograph of the fracture zone in the animal of the experimental group on day 3, where CD34 + cells in the regenerate zone were detected (position 5), immunohistochemical staining for CD34 +, 400 magnification,

Fig.9 is a morphological photograph of the injury zone in the animal of the control group on day 14, single fissile myosattelites are visible (position 6), hematoxylin-eosin staining, 400 magnification,

Figure 10 - morphological photograph of the injury zone in the animal of the experimental group on day 14, multiple fissile myosattelites are visible (position 6), hematoxylin-eosin staining, magnification 400.

The essence of the proposed "Method of stimulating the regeneration of cartilage, bone, muscle tissue" is as follows:

A preparation is prepared that has the property of stimulating the regeneration of cartilage, bone, muscle tissue, based on an aqueous solution of recombinant interferon beta (IFN-β) at a concentration of 1 ∙ 10 ⁵ IU / ml and an aqueous solution of polyethylene glycol with a molecular weight of 6000 (PEG 6000) in concentration of 0.03 ∙ 10 ^-9 mol / ml. Mix the prepared solutions in a ratio of 1: 4, respectively. For this, sterile distilled water is added to a vial containing lyophilized recombinant interferon-beta (IFN-β) to obtain a concentration of 1 ∙ 10 ⁵ U / ml. The vial with the solution mixture is kept for one hour at room temperature, stirring gently. A sample of a polyethylene glycol with a molecular weight of 6000 (PEG 6000) is prepared and dissolved in a separate bottle in distilled water. It is kept at room temperature for 1 hour. The resulting solution is passed through a sterile Minisart porous membrane. The resulting sterile PEG solution is mixed with a solution of interferon-beta.

The prepared product is used to stimulate the regeneration of cartilage, bone, muscle tissue in the treatment of fractures of long bones. To do this, under aseptic conditions, the prepared agent is slowly introduced through the injection needle, consisting of a mixture of aqueous solutions of recombinant interferon beta (IFN-β) and polyethylene glycol. After this, retrograde intramedullary osteosynthesis of a femoral fracture is performed. The wound is sutured in layers.

The inventive "Method of stimulating the regeneration of cartilage, bone, muscle tissue" was tested on laboratory animals (male rats of the Wistar strain weighing 250-300 g, at the age of 6 months). Experimental studies were performed in the scientific department of experimental surgery with vivarium FSBI "NTSRVH" SB RAMP with free access to food and water on a diet that complies with GOST standards. During the study, all bioethical norms of working with experimental animals were carried out according to the order of the Ministry of Health of the USSR dated 08/12/1977, No. 755, as well as the "Rules for work using experimental animals", approved by Order of the USSR Ministry of Health No. 742 of 11/13/1984 The protocol of the experiment was approved by the Committee on Biomedical Ethics of FSBI “NTSRVH” SB RAMS.

Before the experiment, a preparation was prepared that has properties that stimulate the regeneration of cartilage, bone, muscle tissue by the proposed method. For this, the prepared aqueous solutions of recombinant interferon-beta (IFN-β) and high molecular weight polyethylene glycol (PEG 6000) were mixed. A solution of polyethylene glycol (PEG 6000) is prepared at a concentration of 0.03 × 10 ^-9 mol / ml, then mixed with a solution of interferon-beta (IFN-β) at a concentration of 1 × 10 ⁵ U / ml in a ratio of 1: 4, respectively.

For the experiment, recombinant rat interferon beta (IFN-β) was used. The product was lyophilized from a buffered saline solution of citric acid and 125 mM trialose. The contents of the vial are dissolved in 1 ml of sterile distilled water and a concentration of 1 ∙ 10 ⁵ U / ml is obtained. The vial is kept for one hour at room temperature, gently mixing several times. On a torsion balance, a weight of 7.5 mg of polyethylene glycol with a molecular weight of 6000 (PEG 6000) is weighed. Dissolve in a separate bottle in 4 ml of distilled water. It is kept at room temperature for 1 hour, gently stirring several times. The resulting solution was passed through a sterile Minisart porous membrane. The resulting sterile PEG solution is mixed with a solution of interferon-beta, mixed with gentle movements several times. The total volume of the resulting solution is 5 ml. Aliquot the resulting solution into 0.1 ml insulin syringes (1 dose containing 2000 U of interferon-beta).

The regenerating properties of the claimed agent prepared by the proposed method for stimulating the regeneration of cartilage, bone, muscle tissue are investigated.

Experimental animals (Wistar rats) were distributed as follows:

Group 1 - control, animals using an osteotome modeled a transverse fracture of the femur, then performed retrograde intramedullary osteosynthesis with a pin;

Group 2 - experimental, each animal, in addition to intramedullary osteosynthesis with a pin, was injected into the bone marrow canal and surrounding muscle tissue in the fracture zone with a preparation prepared on the basis of rat recombinant IFN-β in a volume of 0.1 ml containing 2000 units of interferon beta (2 ∙ 10 ³ IFN-β).

The operation was performed under intraperitoneal anesthesia (ketamine 50 mg / kg). The surgical field was treated with a solution of primrose. Access to the proximal metadiaphysis of the femur was carried out on the outer surface of the thigh, 2.0 cm long. Using an osteotome, a transverse fracture of the femur was obtained.

Under aseptic conditions, the animals of the experimental group were slowly injected intradermally through an injection needle with a prepared agent consisting of a mixture of aqueous solutions of recombinant interferon beta (IFN-β) and polyethylene glycol, in a volume of 0.1 ml, containing 2000 units of interferon beta (2 ∙ 10 ³ IFN -β). After that, retrograde intramedullary osteosynthesis of a femoral fracture with a pin of 3.5 cm length was performed. The wound was sutured in layers. Removal of skin sutures was performed on the 14th day after the operation.

The experimental animals were taken out of the experiment on the 3rd, 9th, 14th and 35th days after the fracture and administration of the prepared agent in a volume of 0.1 ml containing 2000 IU of recombinant rat interferon beta (2 ∙ 10 ³ IFN-β) and PEG at a concentration of 0, 03 ∙ 10 ^-9 mol / ml. In each breeding period, 5 animals were observed.

The animals were removed from the experiment by intraperitoneal administration of ketamine at a dose of 100 mg / kg. The organocomplex — the hind limb of the rat — was isolated and anatomical preparation was performed, highlighting the defect zone with the surrounding tissues. Material for histological examination was selected by collecting fragments of a limb, including an experimental exposure zone with surrounding tissues. They were fixed with FineFix solution (Milestone, Italy), decalcified. Serial sections were made 3 μm thick, stained with hematoxylin-eosin, magnification 40, immunohistochemical staining was performed on CD34 + (CD34 (ICO 115) mouse monoclonal IgG (Santa Cruz, Cat. N Sc-7324, Lot # C 0909), working dilution 1 : 300).

It was found that on day 3 when applying hematoxylin-eosin staining, no differences were found in the fracture zone in the experimental and control groups: paraossal hematoma, tissue detritus, neutrophilic infiltration were found in all.

On day 9, in all animals of the control group, low to moderate density fibrous callus was formed, the cellular component of which was predominantly represented by fibroblasts. A morphological photograph of the fracture zone in the animal of the control group is presented; fibrous callus is visible (see the appendix to the description of Fig. 1, position 1).

In the experimental group, on day 9, in all cases, high-density fibrous corn was formed, consisting of fibroblasts and fibrocytes. In all animals in the fracture zone, the formation of islets of cartilaginous tissue without signs of enchondral ossification was noted and periosteal foci of osteogenesis were observed. A morphological photograph of the fracture zone in the animal of the experimental group is given, a callus represented by fibrous (position 1), cartilage tissue (position 2) and periosteal callus (position 3) is visible (see the Appendix to the description of figure 2, positions 1, 2, 3 )

On the 14th day, in animals of the control group there is a forming callus, represented by fibrous tissue, which is replaced by cartilage. A morphological photograph of the fracture zone in the animal of the control group is presented, fibrous callus (position 1) with cartilage tissue zones (position 2) is visible (see the appendix to the description, Fig. 3, positions 1, 2).

In the experimental group, on the 14th day, the formation of large zones of cartilaginous tissue, periosteal foci of osteogenesis was noted in all animals. A morphological photograph of the fracture zone in the animal of the experimental group is presented, a small zone of fibrous callus (position 1), an extensive zone of cartilage tissue (position 2), a large area of periosteal callus (position 3) are visible (see the appendix to the description, Fig. 4, positions 1, 2, 3).

On the 35th day in the control group, the regenerate includes chondrogenic tissue, which is replaced by osteogenic tissue with emerging bone beams. A morphological photograph of the fracture zone in the animal of the control group is presented, the remains of the cartilage callus (position 2), the formed bone beams (position 4) are visible (see the appendix to the description, Fig. 5, positions 2, 4).

In the experimental group, on the 35th day, the corn is completely represented by bone tissue with already formed bone beams. A morphological photograph of the fracture zone in the animal of the experimental group is presented, formed bone beams are visible (position 4) (see the appendix to the description, Fig.6, position 4).

We found that progenitor cells (bearing CD34 + receptors) appear in the regenerate zone in the experimental group of animals on the 3rd day, while in the control group of animals they appear only on the 9th day. A morphological photograph of the fracture zone in the animal of the control group on day 9 is shown, where CD34 + cells in the regenerate zone were identified (position 5) (see the appendix to the description, Fig. 7, position 5), immunohistochemical staining on CD34 +, magnification 400. In figure 8 shows a morphological photograph of the fracture zone in the animal of the experimental group on day 3, where CD34 + cells in the regenerate zone were detected (position 5) (see the appendix to the description, Fig. 8, position 5), immunohistochemical staining for CD34 +, magnification 400.

A significant increase in muscle tissue regeneration was observed in the experimental group of animals compared with the control group, which was expressed in increased division of myosattelites in the injury zone in animals of the experimental group (see the appendix to the description, Figs. 9, 10, position 6). The figure 9 shows a morphological photograph of the injury zone in the animal of the control group on day 14, where single fissile myosattelites are visible (position 6), hematoxylin-eosin staining, magnification 400. Figure 10 shows the morphological photograph of the injury zone in the animal of the experimental group on day 14, visible multiple fissile myosattelites (position 6), hematoxylin-eosin staining, 400 magnification.

It was also revealed that the frequency of purulent-inflammatory complications in the experimental group was significantly lower than in the control group (Z = 2.616, p = 0.0089).

Thus, the research results showed that the claimed "Method of stimulating the regeneration of cartilage, bone and muscle tissue" allows, in comparison with known methods, to increase the effectiveness of treatment for fractures of long bones, reduce its invasiveness by preparing funds with the selection of the optimal concentration of drugs used, simplifying the technology stimulation of tissue regeneration in the fracture zone, as well as reduce the frequency of purulent-inflammatory complications.

Sources of information taken into account:

1. Ketlinsky S.A. Cytokines. / S.A. Ketlinsky, A.S. Simbirtsev. - St. Petersburg: Tome, 2008, 549 p.

2. Kalvakolanu D.V. Interferons: cellular and molecular biology of their actions / D.V. Kalvakolanu, EC. Borden // In: Bertion, JR., Editor. Encyclopedia Cancer, 2002, v. 2, p. 511-521.

3. A tool with immunomodulatory, antiviral, antibacterial, antioxidant, membrane-stabilizing, stimulating the regeneration of cartilage and connective tissue properties: US Pat. 2349339 Ros. Federation: IPC A61K 38/21, A61P 37/02, A61P 19/02 / V.V. Malinovskaya, N.V. Vardanyan, V.V. Parfenov; Applicant and patent holder Malinovskaya V.V. - No. 2007126556/15; declared July 12, 2007, publ. 03/20/2009, bull. No. 8, 1 p.

4. Interferon conjugated with polyethylene glycol: patent RU 2433135 C1: IPC C07K 14/52 / C.V. Sheremetyev, V.V. Zverev, S.A. Korovkin, patent holder “FORT Limited Liability Company” - No. 2010140711/10; declared 10/06/2010, publ. 11/10/2011, Bull. No. 31.

5. A method of stimulating reparative osteogenesis in the treatment of fractures: US Pat. 2364361 Ros. Federation: IPC A61B 17/56 / R.Sh. Mirkhaydarov, A.A. Grigoriev, R.K. Urazbakhtin, A.Yu. Ruchko, S.V. Grishanin; Applicant and patent holders R.Sh. Mirkhaydarov, A.A. Grigoriev, R.K. Urazbakhtin, A.Yu. Ruchko, S.V. Grishanina. - No. 2008111519/14; declared 03/25/2008, publ. 08/20/2009, bull. No. 23, 1 p.

6. A method of stimulating reparative osteogenesis: US Pat. RU 2373883: IPC А61В 17/56 / S.P. Mironov, N.P. Omelyanenko, V.K. Ilyina, I.N. Karpov; applicant and patent holders S.P. Mironov, N.P. Omelyanenko, V.K. Ilyina, I.N. Karpov. - No. 2008113737/14; decl. 04/11/2008, publ. 11/27/2009.

7. A method for optimizing reparative osteogenesis: US Pat. 2315 580 Ros. Federation: IPC A61V 17/56 / O.V. Beydik, V.V. Annikov, P.V. Glybochko, V.N. Nikolenko, A.V. Zaretskov, K.K. Levchenko, Wang Kai, D.A. Markov; applicant and patent holder Beidik O.V. - No. 2006102390/14; declared 01/27/2006, publ. 01/27/2008, Bull. No. 3, 1 p.

Claims (1)

A method of stimulating the regeneration of cartilage, bone, muscle tissue, including reposition of a long bone fracture and introducing the prepared agent into the bone marrow channel and surrounding muscle tissue, characterized in that they stimulate tissue regeneration by introducing a mixture of recombinant solutions into the bone marrow channel and surrounding muscle tissue interferon beta (IFN-β) at a concentration of 1 ∙ 10 ⁵ U / ml of polyethylene glycol of molecular weight 6000 (PEG 6000) with a concentration of 0.03 ∙ 10 ^-9 mol / ml in a 1: 4 ratio, respectively, followed by Retrograde intramedullary fixation of long bone fractures.

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