RU2840764C1 - Composition for stimulating regeneration of human connective tissue, method for preparation and use thereof - Google Patents

Abstract

FIELD: medical science.

SUBSTANCE: group of inventions relates to medicine, cell technologies and regenerative medicine and includes a composition for stimulating the regeneration of human connective tissue, comprising suspension of human leukocytes in human blood serum in concentration of 1100-1800×10³ cells/ml, pre-activated with lipopolysaccharide for 8-24 hours in an in vitro culture of platelet granules, cytokines, interleukins, growth factors b-FGF, TGF-b2, VEGF, TDGF and lipopolysaccharide, a method for its preparation and use for stimulating the regeneration of human connective tissue damages.

EFFECT: use of the composition accelerates wound healing, skin growth, vascular and connective tissue growth; reduction of oedema, inflammation and pain syndrome.

3 cl, 5 dwg, 3 ex

Description

Field of technology to which the invention relates

The group of inventions relates to the field of biotechnology and medicine and is a composition for stimulating the regeneration of connective tissue damaged due to burns and/or injuries, a method for obtaining it, as well as a method for treating damaged connective tissue using the said composition. The group of inventions can be used in the treatment of soft connective tissue lesions (mechanical and thermal damage), as well as in the treatment of musculoskeletal injuries (bone fractures) and degenerative-dystrophic changes in the musculoskeletal system and soft tissues (intervertebral hernias, arthrosis of the joints).

State of the art

A composition for regenerating skin and mucous membranes is known, including a culture nutrient medium, including biologically active compounds based on low-molecular peptides and cytokines, used for in vitro cultivation of eukaryotic cells, and conditioned by the products of vital activity and growth factors of eukaryotic cells, secreted by them into the nutrient medium during their cultivation [Patent RU 2455354 C1, published 10.07.2012].

The closest analogue (prototype) of the proposed invention is a composition intended for stimulating wound healing and treating burns, containing a nutrient medium conditioned by a three-dimensional cell culture during in vitro cultivation for a period of up to 14 days [Patent RU 2280459 C2, published 20.01.2005). Cell cultures cultured in three dimensions include stromal and/or parenchymatous cells, including genetically engineered cells. The composition contains the said conditioned culture medium, from which the cells used to condition this medium have been separated. In the composition, the conditioned cell culture medium can be combined with a pharmaceutically acceptable carrier and other compounds. The disadvantages of this known composition are:

- the lack of data on the composition of groups of growth factors, cytokines and steroid hormones included in the conditioned medium, which does not allow us to determine the possibilities of clinical use of the composition and make an informed choice of therapy methods;

- complication of the technological process of creating a composition due to the need for multiple replacement of the culture nutrient medium;

- significant volumes of culture nutrient medium required for cell cultivation processes and conditioning of the culture nutrient medium, which leads to its unjustified consumption and, accordingly, to an increase in the costs of its acquisition;

- a long time to obtain a conditioned environment, the value of which reaches 40 days;

- insufficiently high level of effectiveness of the composition in the treatment process;

- the impossibility of obtaining conditioned media and compositions with increased levels of concentrations of the products of the vital activity of mesenchymal stem cells and growth factors.

Also known is a composition for stimulating cell growth and regeneration, which contains destroyed peripheral blood leukocytes and stem and/or progenitor cells in a conditioned medium for their cultivation with a tissue-specific antigen corresponding to the damaged organ that should be regenerated, and a pharmaceutically acceptable carrier. The culture medium for culturing cells can be any medium for culturing cells that meets the needs of the cultured cells for nutrients, and the duration of the cultivation process can reach 60 days. Conditioning can be carried out by stromal cells, bone marrow cells, parenchymatous cells, liver reserve cells, nerve stem cells, pancreatic stem cells and/or embryonic stem cells [Patent RU2341270 C2, published 10.07.2008; IPC: A61K 35/12].

Thus, the objective of the proposed invention is to increase the efficiency of manufacturing processes and practical application in the treatment process of a composition for stimulating the regeneration of connective tissue.

The achieved technical results of the proposed invention include acceleration of wound healing, skin growth, growth of blood vessels and connective tissue, reduction of swelling, inflammation and pain syndrome.

Disclosure of the essence of the invention

The theoretical premise of the invention was the idea of creating a local depot of autologous living, activated cells in the intradermal layer of the dermis. The effect of cell therapy is mediated both by the presence of cytokines and other biologically active substances synthesized during in vitro incubation in the cell preparation introduced into the intradermal layer of the dermis, and by their continued synthesis in vivo by activated cells after the introduction. An activated blood cell introduced locally into a pathological focus, performing its function, stops pathological inflammation, starts or enhances the natural cascade of healing reactions. Particular efficiency is observed in chronic, long-term damage, when natural cascades of reactions for restoration and regeneration are either weak or blocked by chronic inflammation. Various mediators are used to activate blood cells depending on the purpose of therapy. For example, in the treatment of post-traumatic lesions of joints and periarticular soft tissues, anti-apoptotic mediators (IL6, IL8, TNFa) are used, which stimulate cell proliferation and migration (TGF-b, PDGF, VEGF), and also additionally enhance the synthesis of the extracellular matrix (collagen, elastin and other components of connective tissue) - structural components of ligaments, joints, tendons, which leads to rapid regeneration and restoration of organ function. Restoration of collagen and elastin levels is also widely used for rejuvenation in cosmetology, as well as in anti-age programs.

The main object of action of the proposed composition is Toll receptors. Several types of ligands to this receptor are known. One of them is polyinosinic-polycystidic acid and its salts. However, this synthetic analogue of viral RNA is poorly applicable in clinical practice, as it has a number of side effects and high toxicity.

When the Toll receptor is activated, several transcription factors NF-kB and IRF3 are formed, which in turn activate the transcription of a number of active molecules. The main active substances are the proinflammatory cytokines IL-6, IL-8, TNF, which in addition to their main proinflammatory effect, as it turned out, have an anti-apoptotic effect, keeping proliferating cells viable. These factors also stimulate cell migration and proliferation at the injection site and attract macrophages to the nonspecific inflammation zone. Under the influence of factors (due to increased expression of HIF-1 and VEGF) in the tissue, monocytes are transformed into macrophage cells and participate in regenerative processes. In small quantities, both as a consequence of the fact of nonspecific inflammation and under the influence of Toll receptor activation, growth factors are produced that stimulate angiogenesis, cell proliferation and synthesis of the extracellular matrix. The composition has a pronounced effect of stimulating the synthesis of interferons, which, in turn, have an antiviral effect.

HIF factors play a key role in cell adaptation to oxygen deficiency. The described technology is a powerful stimulator of HIF expression, which leads to activation of genes associated with the adaptive response. For example, HIF promotes the production of erythropoietin (EPO), myoglobin, autonomic neurotransmitters and other metabolic and hormonal factors. The described processes also stimulate autophagic reserves of cells, which accelerates tissue recovery and remodeling, and promotes the regulation of cell growth and survival mechanisms (Fig. 1).

The figure shows a diagram of the sequential processes in tissue after the application of the described technology. The tissue regeneration process occurs due to the inclusion of several mechanisms, including the anti-apoptotic effect of IL-6, IL-8, TNF, growth factors stimulating cell migration and proliferation, as well as antibacterial and antiviral effects.

The proposed composition intended for stimulating the regeneration of human connective tissue contains a suspension of human leukocytes in human blood serum at a concentration of 1100-1800× ¹⁰³ cells/ml, pre-activated with lipopolysaccharide (LPS) for 8-24 hours in an in vitro culture of platelet granules, cytokines, interleukins, growth factors b-FGF, TGF-b2, VEGF, TDGF and lipopolysaccharide. In the present invention, lipopolysaccharide is understood to be the main component of the outer membrane of gram-negative bacteria, preferably isolated from Salmonella typhi bacteria, for example, the drug "Pyrogenal". The said composition is obtained by collecting blood from a patient in a vacuum tube with sodium citrate and in a tube with sodium heparin salt, centrifuging the tube with blood and sodium citrate at 100 g for 15 minutes, followed by collecting platelet-rich plasma from it, which is transferred to a clean vacuum tube without additives. A 10% aqueous solution of calcium chloride is added to the collected plasma at a rate of 50 μl of solution per 1 ml of plasma. The contents are mixed and incubated for 1 hour at a temperature of +18 to +41°C. During the incubation process, a fibrin clot is formed in the tube.

Next, 100-1000 μl of the lipopolysaccharide solution "Pyrogenal" at a concentration of 50 μg / ml are added to the blood in a test tube with sodium heparin, the contents are mixed and incubated for 8 to 24 hours at a temperature of + 18 to + 41 ° C. After incubation, the test tube with blood is centrifuged at 100-200 g for 10 minutes, then plasma with a suspension of leukocytes is collected and mixed with the serum obtained after platelet activation. An increase in temperature in this range and an increase in the incubation time of the test tube with added LPS increases the production of cytokines and growth factors and other biologically active components. After mixing the two components, the test tube can be stored in the refrigerator at a temperature of + 2 to + 8 for 4 hours.

The composition can be used, among other things, to stimulate the regeneration of human connective tissue in cases of injuries that include soft tissue trauma, skin injuries; cut, mine-explosive, bullet, shrapnel or lacerated wounds; fractures, burns.

The method of using the composition of the present invention depends on the type of damage to human connective tissue:

- in case of soft tissue injuries, the wound and its edges are treated with antiseptic solutions. Then the resulting composition is injected with a syringe into the edges of the wound in a total volume of 1-8 ml, depending on the size of the wound. After that, a gauze bandage is applied to the wound and bandaged or a sterile plaster is applied to protect the wound from infection.

- in case of burns, the wound bed and surrounding skin are treated with antiseptic solutions. Then the composition is introduced into the edges of the affected area in a volume of 1-8 ml, depending on the size of the wound. Additional irrigation of damaged tissues with the composition is permissible. After irrigation, you need to wait 5-10 minutes so that the composition is distributed over the surface of the wound and is not absorbed into the bandage immediately after its application.

- in case of bone fractures, the composition is administered directly next to the fracture area in a volume of 1-4 ml; before the injection, the injection site must be treated with an antiseptic solution.

For all the above methods of application, the frequency of administration of one dose of the composition (1-8 ml) is 6-10 times with an interval of 2-3 days between administrations, which is one course of application. The number of courses is from 1 to 4 with an interval between courses from 2 to 4 weeks.

Brief description of the drawings

Fig. 1 Proposed mechanism of regeneration of human connective tissue using the invention;

Fig. 2. Image of soft tissue injury of patient 1 before application of the composition according to example 1 based on autologous serum and autologous leukocyte suspension;

Fig. 3. Image of soft tissue injury of patient 1 one month after the start of using the composition based on autologous serum and autologous leukocyte suspension according to example 1 according to the method according to example 2;

Fig. 4. A wound treated with a known composition based on a conditioned medium according to RU 2280459 C2 on the 3rd day;

Fig. 5. A wound treated with the composition according to item 1 on the 3rd day;

Implementation of the invention

The possibility of implementing the claimed invention is illustrated by the following examples.

Example 1. Obtaining a composition for stimulating the regeneration of human connective tissue

The method for obtaining the composition is a sequence of the following stages:

1) blood collection from the patient into a vacuum test tube containing a 3.2% aqueous solution of sodium citrate in a volume ratio of 9:1 to blood and into another test tube with sodium heparin at a rate of 17 IU per 1 ml of blood.

2) the test tube with blood and sodium citrate is centrifuged at 100-300 g for 15 minutes. After centrifugation, platelet-rich plasma is collected from the test tube and transferred to a clean vacuum test tube without additives. A 10% aqueous solution of calcium chloride is added to the collected plasma at a rate of 50 μl of solution per 1 ml of plasma. The contents are mixed and incubated for 1 hour at a temperature of +18 to +41°C. During the incubation, a fibrin clot forms in the test tube. Then the test tube is centrifuged for 10 minutes at 1500 g. As a result, the fibrin clot settles to the bottom, and serum remains on top, which is collected.

3) 100-1000 μl of LPS "Pyrogenal" solution at a concentration of 50 μg/ml per 1 ml of blood are added to the blood in a test tube with sodium heparin, the contents are mixed and incubated for 8 to 24 hours at a temperature of +18 to +41°C. After incubation, the test tube with blood is centrifuged at 100-200 g for 10 minutes, then plasma with a suspension of leukocytes is collected and mixed with the serum obtained after activation of platelets, in equal proportions. The finished composition can be stored in the refrigerator at a temperature of +2 to +8 for 4 hours.

The concentration of growth factors b-FGF, TGF-b2, VEGF, TDGF in the composition was determined by ELISA, using specific ELISA kits for each type of growth factors. Comparison of growth factor concentrations in the composition was carried out with plasma obtained by taking blood in a test tube with sodium heparin at a rate of 17 IU per 1 ml of blood, centrifuging for 10 minutes at 100-200 g and collecting plasma with a suspension of leukocytes in 5 samples from different patients showed a reliable increase in the amount of growth factors b-FGF, TGF-b2, VEGF in the composition compared to the control plasma. The concentration of VEGF in the control plasma was 196±9.5 pg/ml, and in the composition 324.3±916.8 pg/ml, TGF-b2 - 1.5±0.4 ng/ml and 14.2±2.6 ng/ml, b-FGF - 3.2±0.2 pg/ml and 18.4±4 pg/ml, respectively.

Example 2. Clinical application of the composition

Patient 1. Shrapnel wound of the thigh with massive damage to soft tissues and a fracture of the femur. Upon admission to the hospital, primary surgical treatment of the wounds was performed and the Ilizarov apparatus was installed to fix the femoral fragments. The patient was in the hospital for about a month after admission, regular wound treatments were performed. During this time, the dynamics of restoration of damaged soft tissues was poorly expressed, the thickness and depth of the wound channels remained, and the dynamics of bone callus formation at the fracture site were poorly expressed. After a month of his stay in the hospital, therapy was started using a composition based on a suspension of leukocytes in blood serum obtained according to Example 1 from the autologous blood of Patient 1. Injections of the composition were performed in a total volume of 6 ml, with approximately 0.5 ml injected into the edges of the wound channels and into the fracture area with each puncture. The procedure was performed 2 times a week with an interval of 2-3 days for 4 weeks. The total number of injections was 8. After just one week of therapy, the wound channel lumen began to decrease, the wound edges began to contract, and epithelialization of the wound bed began. The clinical results before and after the use of the composition based on autologous serum and autologous leukocyte suspension are shown in Fig. 2 and Fig. 3. When comparing the results before therapy and after 1 course of therapy shown in Fig. 2 and Fig. 3, respectively, it is evident that after the use of therapy, there is a significant closure of the wound channel and convergence of the edges of the wound field, which indicates an increase in the amount of new connective tissue, the color of the wound changed, it became pinker, which indicates an increase in the vascular network at the site of injury and improved blood supply. When palpating the edges of the wound before the use of therapy, an increased density of soft tissues was noted, after the use of therapy, when palpating, the edges of the wound became softer and more elastic, which indicates a decrease in tissue edema.

Example 3. Comparative study of composition on an animal model

A comparative experiment was conducted on an animal model of the composition presented in RU 2280459 C2 and the composition presented in this invention. The animal model was 6 rabbits of different weights, different ages, different sexes, three males, three females. A skin area was taken from the rabbits and a fibroblast culture was obtained according to the method described in the prototype invention. The fibroblasts were cultured in a closed culture system using a DMEM culture medium with a high glucose content (10% BCS with the addition of 2 mM L-glutamine and 50 mg/ml ascorbic acid) at 37°C in a 5% _CO2 atmosphere with high humidity. After 10 days, the cell culture was removed and fresh medium was added. The cells were cultured for another 4 days. The resulting conditioned medium, exposed to cell and tissue culture for four days (days 10-14), was then collected and concentrated 10-fold in a positive pressure concentrator. A total of 10 ml of 10-fold concentrated conditioned medium was obtained. The final 1-fold concentration of conditioned medium was obtained from 10-fold conditioned medium added to the main medium as a 10% (v/v) solution.

The composition for accelerating regeneration presented in this invention according to claim 1 was obtained by the method according to claim 2 and was manufactured immediately before the beginning of the experiment from autologous blood of rabbits. Surgical incisions no longer than 2 cm were made on 6 rabbits on the back along the spine, 3 on the right and 3 on the left. After making the incision, 2 sutures were applied with non-dissolving threads to bring the edges of the wound together. The operation was performed under anesthesia. The wounds, on one side, were treated with a composition based on a conditioned medium obtained by the method described above, by introducing 2 ml into the edges of each wound in papules of 0.5 ml. The wounds on the other side were treated once with the composition according to example 1 obtained according to example 2 by introducing 2 ml into the edges of each wound in papules of 0.5 ml. A visual assessment of the wounds was made on the 3rd, 7th and 14th days after the operation, and sections of the wound were also removed for histological examination. The results of the visual assessment on day 3 are presented in Fig. 4.5.

According to the results of the visual assessment, it is evident that on the 3rd day, the wounds treated with the composition according to item 1 have less swelling of the edges, are less hyperemic, and are closer together than the group of wounds treated with the composition based on the conditioned medium. This indicates that the signs of inflammation are less pronounced in them and the regeneration processes are faster (Fig. 4 and Fig. 5).

On day 7, rabbit number 4's wounds treated with the composition based on the conditioned medium heal poorly. This is evidenced by swelling around the wound area, an enlarged scab over the wound, which indicates pronounced inflammatory processes and inhibition of healing. At the same time, the group of wounds treated with the composition according to item 1 looks calm, without obvious signs of an inflammatory process. There is no swelling, the edges are close together. The scab is weakly expressed. In rabbit number 3, on day 7, when comparing the groups of wounds, a more pronounced healing process is observed in the group of wounds treated with the composition according to item 1, more closely spaced wound edges, and weakly expressed swelling of the wound edges.

On the 14th day, the signs of inflammation (edema, hyperemia) disappeared in both groups of wounds, which indicates the progress of the healing processes. When comparing the degree of wound healing, it is clear that in the group of wounds treated with the composition according to item 1, the edges of the wounds partially grew together. This is not the case in the second group of wounds. This confirms that in the group of wounds treated with the composition according to item 1, the healing processes occurred faster than in the comparison group.

Results of histological examination.

On day 3, signs of inflammation were observed in the group of wounds treated with the composition based on the conditioned medium. There were foci of lymphoid infiltration. The beginning of the growth of multilayered epithelium was noticeable. In the area of damage to the muscle layer, a large cavity with signs of tissue decay and hemorrhage was observed. On day 7, signs of inflammation persisted, an outgrowth of multilayered epithelium in the form of narrow strands was observed, a cavity with an insignificant amount of lymphoid cells remained in the area of damage to the muscle layer. On day 14, the wound was largely, but not completely, covered by multilayered epithelium, no formation of hair follicles was detected, the cavity in the area of damage to muscle fibers remained, the initial stage of myotube growth was observed.

In the group of wounds treated with the composition according to item 1, already on the 3rd day, a significant growth of the multilayered epithelium was observed, the proliferation of cells in the basal layer of the epidermis and in the cells of the papillary layer increased. No signs of inflammation under the epidermis were observed. On the 7th day, hair follicle outgrowths were formed in the epidermis, the wound lumen was largely closed, partial growth of new muscle fibers was observed, single lymphoid cells and macrophages were observed. On the 14th day, the wound lumen was closed by multilayered epithelium and loose connective tissue under the epidermis layer, the cut of muscle fibers was mostly filled with new muscle fibers.

Based on the results of the histological examination, it can be concluded that for wounds that were treated with the composition according to item 1 throughout the entire observation period of 3-14 days, signs of inflammation were significantly less pronounced, there was a more pronounced growth of epithelium and loose connective tissue, and full growth of new muscle fibers compared to the group of wounds treated with a composition based on a conditioned medium.

From the presented data of visual control of the healing of the surgical wound and histological examination, it can be concluded that the presented composition according to item 1 and obtained according to item 2 has a more pronounced anti-inflammatory effect and an effect that accelerates regeneration.

Claims (3)

1. A composition for stimulating the regeneration of human connective tissue, containing a suspension of human leukocytes in human blood serum at a concentration of 1100-1800× ¹⁰³ cells/ml, pre-activated with lipopolysaccharide for 8-24 hours in an in vitro culture of platelet granules, cytokines, interleukins, growth factors b-FGF, TGF-b2, VEGF, TDGF and lipopolysaccharide. 2. A method for producing a composition according to claim 1, containing a suspension of human leukocytes in human blood serum at a concentration of 1100-1800×10 ³ cells/ml, characterized in that autologous patient plasma obtained after collection in a test tube containing 17 IU sodium heparin per 1 ml of blood, adding 100-1000 μl of a lipopolysaccharide solution at a concentration of 50 μg/ml per 1 ml of blood to the patient's blood sample, followed by incubation for 8-24 hours at a temperature of +18 to +41°C to activate leukocytes in an in vitro culture of platelet granules, cytokines, interleukins, growth factors b-FGF, TGF-b2, VEGF, TDGF and lipopolysaccharide, and centrifugation at 100-200 g, and autologous serum patient, obtained by collecting blood in a test tube with a 3.2% aqueous solution of sodium citrate in a volume ratio of 9:1 to blood, centrifuging at an acceleration of 100-300 g, collecting platelet-rich plasma, adding a 10% aqueous solution of calcium chloride to the plasma at a rate of 50 μl of solution per 1 ml of plasma, followed by incubation of the resulting mixture for 1 hour at a temperature of +18 to +41°C, centrifuging for 10 minutes at 1500 g and collecting serum. 3. Use of the composition according to item 1 for stimulating the regeneration of human connective tissue in case of injuries, including soft tissue injuries, skin injuries; cut, mine-explosive, bullet, shrapnel or lacerated wounds; fractures, burns.