RU2590859C1 - Method of distant hepatocyte regeneration stimulation - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine and biology, particularly concerns hepatocyte regeneration stimulation. For this purpose, under experimental conditions animal is subcutaneously or intramuscularly administered with porous graft in form of calcium phosphate matrix with pore diameter in range of 150-600 mqm, or in form of said matrix with pre-applied syngeneic bone marrow.

EFFECT: method provides high regenerative potential of endogenic hepatocytes throughout long period with lower risk of potential complications and reduction of costs.

1 cl, 2 ex, 2 tbl, 4 dwg

Description

The invention relates to biology and medicine, specifically to regenerative medicine, and for stimulating the regeneration of hepatocytes and can be used to correct structural and functional disorders of the liver, primarily its age-related changes.

The liver in the adult body, as a rule, is at rest, which is accompanied by a non-proliferating state of hepatocytes [Bader A. Method of tissue regeneration: RF patent for the invention No. 2392314 from 06.20.2010]. Physiological regeneration of the liver, in contrast to reparative, is poorly expressed. One of the principal indicators of liver regeneration is the number (fraction) of binuclear hepatocytes (fissile and non-fissile), the number of which is small in the intact liver and, moreover, significantly decreases with age [Severin MV, B.G. Yushkov, Yastrebov A.P. Tissue regeneration under extreme effects on the body. Yekaterinburg: Ural State Medical Institute, 1993. - 187 s - S. 82-91]. There is an inverse proportion when small losses of liver cells are restored slowly, large losses of liver cells are much faster [Severin MV, B.G. Yushkov, Yastrebov A.P. Tissue regeneration under extreme effects on the body. Yekaterinburg: Ural State Medical Institute, 199 .-- 187 s - S. 79; Bader A. Method of tissue regeneration: RF patent for the invention No. 2392314 from 06.20.2010].

Slow subthreshold losses of hepatocytes are not compensated by the body when the process reaches a "critical number" (for the liver this number is 10% of the working hepatocytes), this leads to irreversible processes and death of the body [Severin MV, B.G. Yushkov, Yastrebov A.P. Tissue regeneration under extreme effects on the body. Yekaterinburg: Ural State Medical Institute, 1993. - 187 s - S. 79]. Therefore, the development of methods for stimulating liver regeneration, first of all, its physiological components, is one of the most important socio-economic tasks of anti-aging (regenerative) medicine, aimed at maintaining active longevity in conditions of increasing the average age of the planet's population.

A wide range of drugs is known, biotechnological (hormones, recombinant cell growth factors, cytokines, biologically active substances, etc.) and cellular compositions that can have a stimulating effect on the growth and regeneration of hepatocytes, the functional activity of the liver, especially in case of reparative and pathological organ regeneration.

For example, the effects of dehydrotimine, thymine, metacil, pentoxyl, thyroxine, prednisolone, prolactin, choriogonin, DNA, and products of the regenerating liver have been proven to stimulate liver regeneration [Karagyulyan SR, Svanadze N.L. Strengthening regeneration with extensive liver damage // Surgery. - 1985. - No. 2. - S. 139-143]. Vagosympathetic novocaine blockage of the neurovascular bundles in the neck with mechanical damage to the liver increases the proportion of binuclear hepatocytes, which is a marker of stimulation of reparative regeneration and organ hypertrophy [Tarasko A.D., Ibatullin I.A., Its V.E., Aref'eva A. TO. A method of stimulating reparative regeneration and hypertrophy of the liver when modeling its mechanical damage in the experiment: RF patent for the invention No. 2168336 from 09/08/1999].

Closer to the essence of the invention are methods using biotechnological and cellular compositions and technologies that are used for local or systemic effects on liver regeneration.

One approach is based on the use of complex heterologous tissue extracts, such as extracts from the pituitary or hypothalamus, to induce cell reproduction, such as hepatocytes. However, the use of extracts from animal or human tissues in a laboratory or clinic is problematic due to the possibility of transmission of viral diseases, such as human papillomavirus, spongiform encephalopathy in cattle, swine or sheep viruses. In addition, it is difficult to determine and standardize the quality of the extracts, since it, among other things, depends on the source and cultivation conditions [Bader A. Method for tissue regeneration: RF patent for the invention No. 2392314 from 06.20.2010].

Another approach uses the induction of cell growth by introducing hormones (e.g., growth hormone, STH), growth factors, such as transforming growth factor beta (TGF-beta, TGF-beta), epidermal growth factor (EGF, EGF), vascular endothelial growth factor (VEGF, VEGF) or hepatocyte growth factor (HGF, West Germany), erythropoietin (EPO), thrombopoietin (TPO), granulocyte-macrophage colony stimulating factor (GM-CSF, GM-CSF), granulocyte colony-stimulating , G-KSF) and others [Bader A. Method of tissue regeneration: RF patent n and invention No. 2392314 of 06/20/2010]. They mimic the humoral (paracrine - local and via blood) pathway of the regulatory effect of body cells (e.g., bone marrow) on the regeneration of other cellular systems, including hepatocytes. At the same time, their use in vivo is not without problems because of the pronounced side effects associated with the difficulty of individually selecting a therapeutic dose for each patient and a wide range of cellular and intracellular effects, for example:

1) a fever develops;

2) oncogenes are activated, triggering cell division, but, on the other hand, leading to tumor cell transformation. Moreover, under conditions of liver pathology and physiological aging of the body, mutant cells accumulate, which can be fixed, in particular, by the appearance and accumulation of pathological mitoses [Severin MV, B.G. Yushkov, Yastrebov A.P. Tissue regeneration under extreme effects on the body. Yekaterinburg: Ural State Medical Institute, 1993. - 187 s. - P. 85-91].

From the point of view of cellular technologies, there is a known method for treating chronic diseases, a method for producing a biograft, a biograft for treating any chronic diseases, including fatty liver [Shumakov V.I., Gureev S.V., Onischenko N.A., Temnov A.A. Method for treating chronic illnesses (variants), method for producing a biograft (variants) and a biograft (variants): WO 2007091919 (A1) - 2007-08-16]. Before stem and progenitor cells are taken from the bone marrow, immunocorrection is carried out, cells with tissue-specific antigen are cultured for 7-15 days to obtain a biograft. To obtain tissue-specific antigen, freshly isolated tissue of an organ identical to the damaged is taken. Allogeneic or xenogenic tissue may be used as a source, and biopsy material may be used if possible. The biograft is introduced into an unknown place, which in the experimental model of fatty degeneration of the liver in guinea pigs increases the percentage of binuclear hepatocytes by 5 times.

Known similar to the previous method of production and use of the composition to stimulate growth and cell regeneration [Botin A.S., Onischenko N.A., Temnov A.A. Growth stimulating and cell regenerating composition and a method for the production thereof: WO 2008082323 (A1) - 2008-07-10]. The composition uses a lysate, including stem and progenitor bone marrow cells. The cells themselves can also be included in the composition on a pharmaceutically acceptable carrier or without it. Cells taken from a patient are pre-cultured for up to 60 days with a tissue-specific antigen corresponding to a damaged organ whose cell regeneration needs to be stimulated. The pharmacological composition can be used as cosmetic additives, food additives and additives in animal feed, internal administration, cell culture, pharmaceutical use, as well as to stimulate hair growth. As a result, in the experimental model of fatty degeneration of the liver in guinea pigs, the percentage of binuclear hepatocytes increases 5 times.

The disadvantages of the known methods include:

- 5-7 steps to obtain a biograft or composition, starting with cell collection, indicating the high cost and complexity of the preparatory procedures;

- long-term manipulations with cells outside the body require high-quality specialized equipment and qualified personnel, carry an increased risk of infection of cellular material;

- immunocorrection is not always acceptable for autoimmune and essential liver diseases;

- not indicated the route of introduction of the composition into the body of guinea pigs for liver regeneration. The appointment of a stem cell composition through the gastrointestinal tract is expensive and impractical, since poor assimilation of the introduced material requires the administration of large doses of the composition, the effect is doubtful; parenteral administration of a cell lysate containing intracellular organelles, which are an antigen, in combination with a tissue-specific antigen can provoke the development of allergic and autoimmune diseases;

- the lack of universality of the methods, as in each case a tissue-specific antigen and donor material (autologous, allogeneic, xenogenic) are selected.

Regeneration of liver tissue is one of the most important indicators in surgical hepatology. An important section is the introduction and development of new methods and methods for stimulating regenerative processes in the liver.

Various methods of stimulating liver regeneration are distinguished, such as ligation of the hepatic artery, portal vein branches, hepatophrenicopexy [Dudarev V.A., Fokin D.V. Experimental morphological substantiation of stimulation of liver regeneration // INTERNATIONAL JOURNAL OF EXPERIMENTAL EDUCATION. 2014. No8. C. l12-113; C. 112], denervation of the hepatic artery [P. Mallet-Guy P. Effects de La neurectomien periartere hepatigue sur L, evolution de L, hepatiti toxigue experimentale. - Lyon Hir., 1962, V. 58. - p. 242-258], resection of the liver [Narcissus T.B. Resection of the liver with cirrhosis // Surgery. - 1973. - No. 5. - S. 79-84]. Such traumatic interventions in the body, causing complications in 20% of cases, are unacceptable for stimulating the physiological processes that underlie the regeneration of hepatocytes.

There is a group of methods for treating liver failure, based on the use of "outside the body" - extracorporeal:

- suspensions of isolated xenogenic hepatocytes in the extracorporeal system “auxiliary liver”, through which the blood of the recipient with the affected liver is perfused [Shumakov V.I. and coauthors. Essays on the physiological problems of transplantology and the use of artificial organs // Tula. - Reporters. - 1998. - p. 343-362];

- microfragments of liver tissue [Soloviev V.V., Onishchenko N.A., Akatov B.C., Lezhnev E.I. Functional activity of hepatocytes in liver fragments in vitro: dependence on the size of the fragments and the duration of their cultivation. // Bull. Expert. Biol. and Med., - 1997, - No. 10, - p. 406-408];

- isolated hepatocytes with microcarriers (for example, cytodex-3) that are pre-coated with collagen [Dimetriou A.A., Rozga J., Podesta L. Early clinical experience with a hybrid bioartificial liver // Scan. J. gastroenterol. - 1995. - 30. - Suppl. 208. - p. 111-117; Chen S., Eguchi S. Watanabe Hepatic support strategies // Transplant. Proc. - 1996. - 28, No. 4. - p. 2036-2038];

- extracorporeal systems filled with collagen gel with hepatocytes [Naka S., Takeshita K., Yamamoto T. Bioartificial liver support system using porcine hepatocytes entrapped in a three-dimensional hollow fiber module with collagen gel: an evaluation in the swine acute liver failure model // Artif. Organs. - 1999. - V. 23. - R. 822-828].

The main disadvantages of methods using hepatocytes outside the body (extracorporeally) for the correction of liver failure are:

- short life of isolated hepatocytes (no more than 1-2 days);

- the need to regularly conduct sessions connecting to perfusion systems "bioartificial liver";

- high cost.

So, to stimulate liver regeneration in case of fibrotic changes of various origins on the model of liver cirrhosis in the experiment, the drug EMBRYOBLASTE is injected into the liver tissue with a needle-injector to a depth of 0.1 cm. As the authors note, the drug causes an increase in the volume of hepatocytes (hypertrophy) [Dudarev V.A., Kirgizov I.V., Yakimova S.I., Fokin D.V., Vorobyeva E.L., Skladneva V.O. A method of stimulating liver regeneration with fibrotic changes in the experiment: RF patent for the invention No. 2455701 from 10.07.2012]. Data on the stimulation of hepatocyte regeneration are not presented.

To stimulate the regeneration of hepatocytes, a course (up to 5 times) administration of dispersed Alloplant biomaterial is carried out by means of endoscopic cannulation of the umbilical vein [Galimov OV, Nurtdinov MA, Shumkin AM, Ziangirov RA, Khafizov N.Kh., Khafizov T.N., Timerbulatov M.V., Garifullin B.M., Senderovich E.I., Bulgakov V.R., Ziganshin I.M. A method for the treatment of toxic hepatitis and cirrhosis of the liver: RF patent for the invention No. 2205647 from 06/10/2003].

To stimulate regeneration, autologous transplantation of fragments of the liver is carried out, which, however, die if the hepatocytes produced by poisonous bile are not removed [Severin MV, B.G. Yushkov, Yastrebov A.P. Tissue regeneration under extreme effects on the body. Yekaterinburg: Ural State Medical Institute, 1993. - 187 s - S. 70-71]. The creation of a tap (anastomoses) is a microsurgical operation that requires highly skilled specialists. There is a resorption of the base part of the transplanted material. Transplanted tissue fragments have low functional and regenerative activity.

There is a method consisting in the introduction of xenogenic hepatocytes: subcutaneously into the anterior abdominal wall before surgery and under the capsule of the kidney during partial resection of the liver. The method increases liver regeneration [Chikoteev S.P., Plekhanov A.N., Tovarshinov A.I., Goldberg O.A., Lepekhova S.A., Kornilov N.G. The method of post-resection liver regeneration in the experiment: RF patent for the invention No. 2232550 from 07.20.2004].

A method for the treatment of chronic diffuse liver diseases is shown, which involves the injection of a suspension of fetal cells, human tissues, moreover, the introduction is carried out subcutaneously at 4-5 points of the anterior abdominal wall, and the course contains 2-4 injections [Transplantation of fetal tissues and human cells / ed. G.T. Sukhikh.- M., International Institute of Biological Medicine, 1996, pp. 84-86]. An alternative is the intraperitoneal administration of a suspension of fetal cells of the liver and / or spleen and / or thymus [Kurbatova GR, Giniatullin R.U., Kozel A.I., Ignatieva E.N. A method for the pathogenetic therapy of chronic liver diseases: RF Patent for the invention No. 2203675 of 05/10/2003]. However, cells that are foreign in their antigens in the abdominal cavity activate resident macrophages, which quickly utilize foreign cells and are capable of provoking the development of inflammation (peritonitis), which was confirmed by the applicants.

The experiments performed by the authors on mice showed that subcutaneous administration of even syngeneic (from animals of the same line) hepatocytes using a substrate is ineffective for long-term stimulation of liver regeneration, since 45 days after implantation, they die and are completely utilized by macrophages. On preparations instead of liver cells, hemosiderophages, blood vessels, connective and adipose tissues that replace liver tissue are found (Fig. 1).

A known method of treating liver failure using a polymer matrix, which is used as a gel and allogeneic donor cells. A gel with cellular material is transplanted into the liver parenchyma and / or mesentery of the small intestine. Immediately after the transplantation of allogeneic liver cells and bone marrow progenitor cells, immunosuppressants are prescribed on a gel in a prophylactic dose, which allows to increase the survival time of cells and enhance their proliferation [Gauthier SV, Shagidulin M.Yu., Onishchenko NA, Krasheninnikov M .E., Sevastyanov V.I. Method and transplant for the treatment of liver failure: RF patent for the invention No. 2425647 from 08/10/2011].

Closest to the proposed is a method of treating liver failure [Gauthier S.V., Shagidulin M.Yu., Onishchenko N.A., Krasheninnikov M.E., Sevastyanov V.I. Method and transplant for the treatment of liver failure: RF patent for the invention No. 2425648 of 08/10/2011], using autologous progenitor bone marrow cells previously cultured in vitro for 7 days. Autologous liver cells are also taken. Autologous cells of the liver and progenitor cells of the bone marrow are co-cultured for 2-3 days. Only after this, a mixture of autologous liver cells and bone marrow progenitor cells is immobilized onto a carrier - a biodegradable polymer three-dimensional matrix. After this, the carrier is transplanted with cells, introducing it into the mesentery of the small intestine. After transplantation of autologous liver cells and bone marrow progenitor cells, anticoagulants and antiplatelet agents are prescribed in a prophylactic dose on the matrix.

However, the above method is not effective enough, the use of xenogenic and fetal material is undesirable.

The disadvantages of using known surgical methods, including the prototype, include:

- the high cost and complexity of preparatory procedures outside the body (up to 7-10 stages of cell manipulation), requiring high-quality specialized equipment and qualified personnel, an increased risk of infection of cellular material;

- inflammatory reaction to the transplant. Polymeric materials undergo biodegradation caused by giant multinucleated foreign body cells [Biomaterials Science: an introduction to Materials in Medicine / ed. by BD Ratner, AS Hoffman, FJ Schoen, JE Lemons. - 2 ^nd ed. - Elsevier Inc., 2004. P. 296-304; Khlusov I.A., Nechaev KA, Dvornichenko MB, Khlusova M.Yu., Saprina T.V., Tverdokhlebov S.I. The effect of hybrid implants based on a three-dimensional polymer matrix on the aging dynamics of leukemic mice // Biotechnosphere. 2010. No. 5-6 (11-12). S. 15-19], which contribute to the development of chronic granulomatous inflammation [Biomaterials Science: an introduction to Materials in Medicine / ed. by BD Ratner, AS Hoffman, FJ Schoen, JE Lemons. - 2 ^nd ed. - Elsevier Inc., 2004. P. 296-304];

- low survival rate of the introduced hepatocytes or other cells due to circulatory disorders in the transplant, especially against the background of liver fibrosis;

- the use of pharmacological preparations (anticoagulants and antiplatelet agents) that increase the load on the recovering liver;

- the invasiveness of abdominal surgery, endoscopic or laparoscopic transplantation into the mesentery of the small intestine or liver parenchyma, the possibility of complications (bleeding, thrombosis, suppuration, the risk of tumor transformation, etc.), the need to navigate to deliver the transplant (under the supervision of an ultrasound or endoscope), which determines the application of the method only in specialized departments involved in the treatment and correction of liver failure, with the involvement of highly qualified specialists;

- correction of liver failure due to the proliferation and functioning of hepatocytes introduced from the outside (replacement therapy), while stimulation of hepatocyte regeneration in the damaged liver has not been proven;

- the geometrical parameters of the used matrix carriers of cells and preparations are practically not presented, although numerous facts are known in the literature of a significant influence of the characteristics of carriers, for example, their porosity parameters, on the vital activity of cells and tissues [Gauthier O., Bouler J.-M., Aguado E. e.a. Macroporous biphasic calcium phosphate ceramics: influence macropore diameter and macroporosity percentage on bone ingrowth // Biomaterials. 1998. V. 19 (1-3). P. 133-139; Sous M., Bareille R., Rouais F. et al. Cellular biocompatibility and resistance to compression of macroporous beta-tricalcium phosphate ceramics // Biomaterials. 1998. V. 19. R. 2147-2153].

Such disadvantages are deprived of the proposed method for distant stimulation of hepatocyte regeneration.

A new technical result is an increase in the regenerative potential of endogenous hepatocytes over a long period of time, an expansion in the scope of application, a decrease in the risk of possible complications, an increase in usability and cost reduction.

To achieve a new technical result in a method for distantly stimulating the regeneration of hepatocytes, including the introduction of a graft into the body, a graft is introduced subcutaneously, which includes a calcium phosphate matrix having a porous structure with a pore diameter of the material in the range of 150-600 μm, bearing a pre-applied syngeneous bone marrow.

Also, in the absence of the possibility of collecting cellular material, a graft is introduced subcutaneously without previously applied bone marrow.

A graft made in the form of a parallelepiped or cylinder is also introduced.

For distant stimulation of hepatocyte regeneration, the method is used for the first time.

The method is as follows

1) A matrix made of calcium phosphates with a pore diameter of the material in the range from 150 to 600 μm is used as a transplant matrix. The most optimal is the geometric shape of the matrix, for example in the form of a parallelepiped or cylinder, with dimensions suitable for subcutaneous or intramuscular minimally invasive administration through a skin incision, for example, a trocar. The choice of technical parameters (pore diameter) is based on maintaining strength with a rather significant porosity of the graft matrix material.

2) The bone marrow is isolated from bones (femur, tibia, ileum, sternum, etc.) by known methods (aspiration, trepanobiopsy, sternal puncture, for bone operations, etc.), injected into the porous structure of the material (syringe, automatic dispenser, etc.), if possible, are cultured for no more than 1 hour at 35-37 ° C in a synthetic nutrient medium for cell culture (for example, DMEM, MEM, RPMI-1640, F12, etc.) [Introduction to cell culture methods, bioengineering of organs and tissues / Ed. V.V. Novitsky, V.P. Shakhova, I.A. Khlusova. - Tomsk: STT, 2004 .-- 386 s .-- S. 37-40; S. 141-143; from. 297-300].

3) the transplant is administered to the experimental animal in the form of a carrier matrix pre-saturated with bone marrow cells, or as a carrier matrix, subcutaneously (intramuscularly), by any of the known less traumatic methods (trocar, small incision) under local or systemic anesthesia.

The proposed method is based on the analysis of the results of experimental studies. The essence of the method is illustrated by the following examples.

Example 1

A graft was made - parallelepipeds with a linear size (5 × 5 × 1 mm ³ ), pressed from the powder by a shaped method at a pressure of 130 kilograms of force per 1 square centimeter, annealed at 600 ° C. The phase composition of the starting powder corresponded to the formula of hydroxylapatite Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ with carboxyl groups. To obtain pores of various diameters in the parallelepipeds (in the range of 150-600 μm), hydroxylapatite particles of the corresponding linear size were used. The bulk porosity of the material was 50-70%. [Karlov AV, Khlusov IA Mikrostrukturische charakteristiken der poren, die osteoinduktiven eigenschaften der kalziumphosphatkeramik bilden // Biomedizinische Technik. - 2000. - Band 45. - Erganzungsband 1. - P. 123-124]. Porous samples consisted of calcium phosphates, mainly hydroxylapatite and tricalcium phosphate, mixed with carboxyl groups, sodium and chlorine, which corresponds to the natural minerals of bone tissue. Table 1 presents data on the distribution of calcium phosphate samples with different linear pore sizes in the dynamics of the experiment, M ± m, where M is the average value, m is the error of the mean.

CBA / CaLac mice under ether anesthesia were injected subcutaneously through a midline skin incision with 1 parallelepiped graft. Before the animals were introduced into the pores of the substrates, under suspension aseptic conditions, a suspension of syngeneous bone marrow isolated from the femur (tibia) of the mice was applied according to the known method [Shakhov V.P., Khlusov I.A., Dambaev G.Ts., Zaitsev K.V. , Egorova A.B., Shakhova S.S., Zagrebin L.V., Volgushev S.A. Introduction to cell culture methods, bioengineering of organs and tissues / Ed. V.V. Novitsky, V.P. Shakhova, I.A. Khlusova. - Tomsk: STT, 2004 .-- 386 p. - S. 141-143]. Bone marrow served as a source of stem cells and growth factors. For cell adhesion, the substrates were cultured for 45 minutes in DMEM / F12 culture medium under aseptic conditions before administration to animals.

Used 5 mice in the control and experimental groups at each point of the experiment. False-operated group consisted of 8 animals.

After 1-6 weeks, the animals were killed by ether anesthesia, parallelepiped-shaped grafts were removed. Part of the liver was taken for histological analysis by light microscopy of thin sections (thickness less than 10 μm). The grafts were fixed in 10% neutral formalin, decalcified, then the grafts and parts of the liver were embedded with paraffin and thin (10 μm) sections were performed perpendicular to the surface of the grafts, stained with hematoxylin-eosin according to the standard method. The results were expressed as mean and error of the mean (M ± m) or as median (Me), first (Q1) and third quartile (Q3).

In histological sections of the grafts, bone marrow and / or bone growth was noted (Fig. 2). Slices of the liver were photographed with a resolution of 8 megapixels. Regenerative processes were quantified using computer morphometry of liver sections in 10 digital micrographs of various fields of view [G. Avtandilov. Diagnostic medical ploidometry. M .: Medicine, 2006. - 192 s]. Counted the percentage of binuclear hepatocytes of the total number of liver cells in the field of view.

The results showed that with subcutaneous injection of grafts in the pores of the calcium phosphate matrix, even without previously applied bone marrow, the development of vascularized connective tissue was observed, to which sections of muscle tissue were adjacent (Fig. 2a). In a porous material seeded with ex vivo bone marrow, sections of coarse fiber with lacunae filled with red bone marrow grow in the pores (Fig. 2b).

A study of the regenerative activity of the liver (Fig. 3, which shows the percentage of binuclear hepatocytes in the liver of mice in the dynamics of regeneration after implantation of porous calcium phosphate material (control) bearing bone marrow (experiment)) showed that the administration of calcium phosphate porous matrix without bone marrow (control) caused a statistically significant (P <0.05 according to Student t-test) increase (up to 128-178% of the initial level) of the indicator at weeks 1-4 after the operation (in the range of the pore diameter of the material 150-600 microns). The addition of bone marrow (experience) led to an increased level (up to 127-203% of the initial, P <0.05 according to the Student t-test) of binuclear hepatocytes throughout the entire 6 weeks of the study (P <0.05 according to the Wilcoxon T-test) in comparison with both the initial value and the performance of control mice.

Thus, against the background of false-operated animals, minimally invasive subcutaneous injection of grafts made of porous calcium phosphate matrices with pore diameters in the range of 150-600 μm causes a distant (distant) regulatory effect on the recipient's liver, leading to long-term stimulation of hepatocyte regeneration, one of the mechanisms of which there may be regeneration of the capillary bed not only at the site of implantation, but also in the liver tissue. Also, pre-applied donor bone marrow enhances and prolongs local regenerative processes, which leads to the formation of a bone / bone marrow system with rich blood supply characteristic of these tissues. At the same time, the distant stimulating effect on the regeneration of hepatocytes significantly increases, in amplitude and duration of action.

To test the operation of the polymeric carrier materials indicated in the prototype (polylactides widely used in medical implantation practice) under the conditions of subcutaneous implantation, the experiment described in Example 2 was carried out.

Example 2

We applied polylactide matrices (PLA) with linear dimensions of 15 × 15 mm and a thickness of not more than 1 mm, with comparable porosity, prepared according to [Tverdokhlebov SI, Stankevich K.S., Bolbasov E.N., Khlusov I.A., Kulagina IV , Zaytsev K.V. Nonwoven Polylactide Scaffolds Obtained by Solution Blow Spinning and the In Vitro Degradation Dynamics // Advanced Materials Research. - 2014 - Vol. 872. - p. 257-263]. Each group had 9 matrices. An in vivo experimental study was performed on 27 sexually mature white Wistar male rats weighing 200 g contained in standard vivarium conditions. Under ether anesthesia, the rats were injected with 1 transplant made of polymer (PLA), subcutaneously, in the abdomen, through a skin incision according to a similar scheme, as described in Example 1. As a control, a group (9 animals) of false-operated rats, which were performed midline skin incision without the introduction of substrates (Table 2 - The number of binuclear hepatocytes in Wistar rats 60 days after subcutaneous implantation of polymer substrates from polylactic acid (PLA control) with bone marrow (PLA experiment), Me (Q1-Q3) Note: n - number of calculated fields vision with computer morphometry).

At the end of the experiment (60 days after transplantation), the rats were withdrawn from the experiment under ether anesthesia. Preparation of liver preparations and their quantitative morphometry were performed as shown in Example 1. The number of binuclear hepatocytes in the field of view was calculated.

The results showed that subcutaneous implantation of polylactic acid transplants, with and without bone marrow, did not cause visible morphological changes in the liver associated with destructive processes. At the same time, the number of binuclear hepatocytes in the control and experimental groups also did not statistically significantly increase (Table 2).

So, with subcutaneous administration, it is transplants made in the form of a porous calcium phosphate matrix that carry pre-applied bone marrow (or without it) that contribute to distant, long-term stimulation of hepatocyte regeneration in the recipient's liver.

The duration of the regenerative effect of 1 procedure for the introduction of a graft made of calcium phosphate material from 4 to 6 weeks in mice when calculated through the average life expectancy (70 years in humans and 2-3 years in mice) can be up to 210 weeks in humans . At the same time, versatility, efficiency and ease of use are achieved.

The proposed method has low invasiveness, creates adequate conditions for the growth of blood vessels due to the structure of the graft, the technical parameters of its structure, which prolongs the adequate life conditions of the planted cells; conditions for the proliferation, differentiation and maturation of stromal and hematopoietic cells of the bone marrow, the formation of bone and hematopoietic tissues.

The use of autologous (syngeneic) cells of adult donors prevents unwanted activation of the immune system, allows this system to have a long bioregulatory effect in the body. The present invention does not use tissue and / or cellular material of human embryos.

The use of bone marrow, which in the process of remodeling and reparative regeneration (donor stromal cells remain on the matrix, the stem hematopoietic cells of the recipient through the growing blood vessels gradually replace the dying hematopoietic cells of the transplanted bone marrow) helps to establish a system of direct and feedback connections with the host organism, due to which hepatocyte regeneration becomes controlled.

The choice of bone marrow was determined by the following facts:

- high content of stem cells;

- resistance to hypoxia, the ability of cells to survive until the moment when the blood vessels grow into the transplant;

- high activity of regenerative processes (rapidly proliferating cell system) and the ability to remodeling. Our preliminary experiments showed that, on a similar matrix, liver cells, unlike bone marrow, do not survive with subcutaneous injection (Fig. 1);

- active secretion of numerous biologically active substances and cytokines that stimulate the regeneration of various cells, including hepatocytes (described above).

The choice of substrate (matrix) is determined by the following facts

- biocompatibility, since calcium phosphates are similar to those in bone tissue;

- multifunctionality (performs both matrix functions and stimulates bone marrow regeneration);

- sufficient mechanical strength for subcutaneous use;

- a porous structure that provides neovascularization of the implant structure, which promotes the growth of cells in the pores of the material ("in-growth"), protects cells from mechanical damage and rinsing from the material, activating their regeneration;

- the possibility of sterilization by standard methods without changing the medical-technical properties of the material;

- the ability to gradual biodegradation;

- calcium phosphates are approved for clinical use.

Also, the proposed method can be carried out using trocars of various diameters for subcutaneous (intramuscular) introduction of transplants, which makes it easy to apply the developed method in clinical practice, since the majority of surgeons and traumatologists own the trocar, which expands the scope, reduces the risk of possible complications, increases the usability and reduces costs.

The proposed method has low invasiveness, creates adequate conditions for the growth of blood vessels due to the structure of the graft, the technical parameters of its structure, which prolongs the adequate life conditions of the planted cells; conditions for the proliferation, differentiation and maturation of stromal and hematopoietic cells of the bone marrow, the formation of bone and hematopoietic tissues.

Thus, the proposed method is promising for implementation in clinical practice.

application

FIG. 1 Substitution of donor hepatocytes on the calcium phosphate matrix with connective and adipose tissues 45 days after subcutaneous administration. Hematoxylin-eosin stain. Increase 100.

Fig 2 - The growth of connective tissue (a), bone and bone marrow (b) in the pores of the grafts. Here: 1 - artificial material; 2 - connective tissue; 3 - bone tissue with bone marrow; 4 - muscle tissue. Hematoxylin-eosin stain. Increase 100.

Fig 3 - Percentage of binuclear hepatocytes in the liver of mice in the dynamics of regeneration after implantation of a porous calcium phosphate matrix material (control) carrying bone marrow (experiment).

Table 1 - Distribution of calcium phosphate matrices with different linear pore sizes in the dynamics of the experiment, M ± m

Table 2 - The number of binuclear hepatocytes in Wistar rats 60 days after subcutaneous implantation of polymer substrates from polylactic acid (PLA control) with bone marrow (PLA experiment), Me (Q1-Q3)

Claims (2)

1. The method of distant stimulation of hepatocyte regeneration in an experiment, characterized in that the animal is injected subcutaneously or intramuscularly with a graft in the form of a calcium phosphate matrix having a porous structure with a pore diameter in the range of 150-600 μm, or in the form of the specified matrix with pre-applied syngeneous bone marrow. 2. The method according to p. 1, characterized in that the transplant is introduced, made in the form of a parallelepiped or cylinder.

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