RU2597085C2 - Implanted matrix material for regenerative medicine and method of its production (versions) - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, particularly to a new matrix material for tissue bioengineering and regenerative medicine and methods of its production. Matrix material is designed based on vegetal polysaccharide - pectin with etherification degree of not more than 50% and proteins of intracellular matrix-collagens I and IV types, which contains said components in the following concentrations: 0.5-2.0 wt%, 0.1-1.5 wt% and 0.01-0.5 wt%, respectively. Method of production of the implanted matrix material for regenerative medicine in the form of hydrogel involves mixing at a temperature of 5 °C max of calcium acetate solution of type I collagen to final concentration of 0.1-1.5 wt% with type IV collagen solution to the final concentration 0.01-0.1 wt%, then the produced mixture is mixed with previously prepared gel formation initiator, including calcium chloride solution in the concentration, providing a gel forming process, non-toxic buffer system in amount sufficient for neutralisation of the mixture, and sodium chloride solution up to physiological concentration; then the pectin solution is introduced with degree of esterification of 50% max to the final concentration 0.5-2.0 wt% and the gel is formed by temperature rise up to physiological. Invention also relates to a method of producing implanted matrix material for regenerative medicine in the form of hydrogel, which involves mixing at a temperature of 5 °C max of calcium acetate solution of type I collagen to final concentration of 0.1-1.5 wt% with IV type collagen solution to the final concentration 0.01-0.1 wt%, then the produced mixture is neutralised, stabilised by the non-toxic buffer system and the sodium chloride is added up to physiological concentration then the pectin solution with etherification degree of not more than 50 % to the final concentration 0.5-2.0 wt% is added, to obtain a fluid composition, from which the gel is formed.

EFFECT: invention enables to obtain a biocompatible implantable materials with a low rate of biodegradation and ability to stimulate regenerative process, in particular, recovery of nerve conductors after a brain injury.

10 cl, 8 dwg

Description

The invention relates to medicine, in particular to a new matrix material for tissue bioengineering and regenerative medicine and methods for its preparation. The main field of application of the material is reconstructive therapy of injuries of the nervous system and pathologies accompanied by neurodegeneration. The material can also be used for reconstructive therapy of injuries of other organs and tissues.

The analogues of the present invention are various biocompatible polymeric materials for tissue engineering and reconstructive therapy. Among analogues, inventions protected by RF patents No. 2188206, IPC C07K 14/78, C07K 1/36, A61K 38/39, A61P 9/08, publ. 2002, No. 22449462, IPC A61K 38/39, A61K 35/12, publ. 2005, No. 2321597, IPC С08В 37/00, С08L 5/00, A61K 31/715, A61K 31/724, publ. 2008 year

Some of these inventions are based on the creation of a biocompatible matrix based on components represented exclusively by various forms of collagen — key extracellular matrix proteins (patents No. 2188206 and No. 22449462). Such materials, despite the demonstrated repair properties, have a number of significant drawbacks. One of the disadvantages of materials based on various types of collagen is their high rate of biodegradation in the body. In the technical solution, protected by RF patent No. 22949462, it is stated that the obtained heterogeneous matrix from collagen preparations of two classes of vertebrate animals, presented as a two-phase polymer system (solid spheres in a solution of denatured collagen), according to the description of the invention, has an adjustable rate of degradation in the body. However, neither in the description of the technical solution, nor in the examples cited by the inventors, it is indicated how the known problem of rapid biodegradation of implantable collagen was solved. The inventors also did not demonstrate the results of experiments that allow to evaluate the rate of biodegradation of this material. Neither in the patent description, nor in the scientific publications of the inventors in the public domain, it is indicated about the use of methodological approaches to assess the rate of biodegradation of collagen in the field of implantation. While from the well-known technical solutions described in the scientific literature, the rate of collagen degradation can be reduced by introducing covalent crosslinks between the polymer chains, as well as the introduction of collagen in combination with slowly degrading polymers (such as alginate, chitosan, agarose). These methods are the only ones described in the scientific and technical literature. Another disadvantage of collagen-based materials is the lack of ability to limit the formation of gliomesodermal scar and dense cysts that occur during the natural repair process after traumatic brain damage due to migration and adhesion of neuroglia and connective tissue cells. This disadvantage of collagen materials is associated with high adhesive properties in relation to neuroglia cells and connective tissue. Although the structures of the forming scar contribute to the restoration of brain integrity, they nevertheless form an insurmountable barrier to the growth and regeneration of nerve cell processes.

Known matrix gel materials based on hyaluronic acid - a carbohydrate that forms the amorphous substance of the extracellular matrix of animals. Such gels support the viability and axonal growth of chicken dorsal ganglion neurons (Horn EM, Beaumont M., Shu XZ et al. Influence of cross-linked hyaluronic acid hydrogels on neurite outgrowth and recovery from spinal cord injury // Journal of Neurosurgery-Spine. 2007 . V. 6. P. 133-140).

However, the implantation of these hyaluronic gels in the area of spinal cord injury in rats does not contribute to the regeneration of the latter. The introduction of a hyaluronic gel into the damaged area of the rat cerebral cortex inhibits the formation of a glial scar, allows for cell migration inside the gel, but does not support axonal growth (Cui FZ, Tian WM, Hou SP, et al. Hyaluronic acid hydrogel immobilized with RGD peptides for brain tissue engineering // Journal of Material Science: Materials in Medicine. 2006. Vol.17. P. 1393-1401). The same gels, but modified with immobilized RGD peptides, do not have this drawback. Not only various cells penetrate into such gels, but regenerating axons also actively grow.

Thus, without the introduction of additional components or chemical modifications, hyaluronate does not contribute to the regeneration of the nervous system. Its other drawback, as well as other natural materials of animal origin, is its rapid biodegradation. Gels consisting only of hyaluronic acid are completely degraded by subcutaneous implantation after 2 weeks (Hahn MS, Teply BA, Stevens MM et al. Collagen composite hydrogels for vocal fold lamina propria restoration // Biomaterials. 2006. Vol.27. P. 1104-1109).

Matrices based on alginates - polysaccharides obtained from brown algae are known. Alginic acid polymers easily form highly hydrophilic, immunologically inert and biocompatible gels when calcium ions are added to them. These gels, unlike gels based on hyaluronic acid, biodegrade slowly in mammals, but when they are implanted in the area of the brain injury, they do not provide axon growth and, consequently, nerve pathway regeneration. To impart neuroregenerative properties to such materials, porous sponges are obtained from gels by freeze-drying the material (Suzuki K., Suzuki Y., Ohnishi K. et al. Regeneration of transected spinal cord in young adult rats using freeze-dried alginate gel // Neuroreport. 1999. Vol.10. P. 2891-2894). Only in this form did alginate-based material stimulate axon growth in the damaged spinal cord of rats, however, researchers were not able to achieve a significant neuroreparative effect that would significantly restore the motor activity of animals, recorded on a standard scale for assessing recovery of motor activity. Another disadvantage of alginates is their low swelling and dissolution rates, which complicates their use in the procedures for obtaining matrix materials.

The closest technical solution in terms of the number of essential features is an implantable porous matrix for regenerative medicine, formed from a biocompatible polymer or polymer mixture, in particular, plant polysaccharides and extracellular matrix proteins are used for this. The formation of the matrix is carried out by compaction of polymer particles and particles of sodium chloride using compression. Then, particles of sodium chloride are removed from the pressed polymer by dissolution. In one embodiment of the matrix, after removal of sodium chloride from it, the surface is covered with extracellular matrix proteins, including type I and IV collagens (Cl. RF No. 2392287, IPC C08J 9/26, A61L 27/56, publ. 2010).

The disadvantages of this matrix include:

- it is not intended for use as a consolidating matrix for reconstructive and regenerative therapy of brain injuries, since it does not impede the formation of a dense gliomesodermal scar and cysts with a dense membrane in the area of traumatic brain damage;

- high porosity of the matrix, which after implantation provides for rapid penetration of cells with high migration and remodeling matrix ability into its structure, which is undesirable, since it leads to a high biodegradation rate of the implant, which is destroyed before the slow processes of restoration of damaged tissues undergo neuroregeneration ;

- the mechanical properties of a rigid porous matrix mimic the natural extracellular matrix of soft tissues worse than that of a hydrogel;

- implantation of the matrix is possible only through complex surgical operations involving intact areas of the body, since tissue dissection procedures are necessary to obtain an open surgical field;

- the complexity of the method of preparation of the matrix, especially in cases of obtaining a composite matrix, since it involves pressing procedures, cycles of washing with a solvent and applying other components of the composition to prepared porous surfaces, which makes it impossible to inject low-traumatic introduction of the material and the formation of the matrix directly in the implantation site.

The objective of this group of inventions is to create a new matrix material for regenerative medicine, intended, in particular, for reconstructive therapy of brain injuries, which provides regeneration of nerve conductors, has a low biodegradation rate in mammals, comparable to the rate of regeneration, and is able to limit the formation of dense scars and large cysts in the focus of tissue remodeling.

The problem is solved by creating a new implantable matrix material for regenerative medicine, based on a biocompatible polymer mixture of plant polysaccharide and extracellular matrix proteins of type I and IV collagen, in which according to the invention pectin is used as a plant polysaccharide with an esterification degree of not more than 50%, and the final the concentration of components in the matrix material are in the range, weight. %:

pectin with an esterification degree of not more than 50% - 0.5-2.0,

type I collagen - 0.1-1.5,

type IV collagen - 0.01-0.5.

In addition to traditional collagen IV preparations, the preparation of its NCl hexamers is also used in the implantable matrix material.

The use of pectin with an esterification degree of not more than 50% makes it possible to obtain stable matrices in the form of hydrogels that are easily implantable in the area of traumatic injuries, which can be effectively colonized by the cells of the regenerating region and stimulate the regeneration of nerve conductors. The use of pectin in the composition with extracellular matrix proteins gives the material a low biodegradation rate in the body and prevents the formation of dense scars, since it reduces the adhesion properties and targeting of cells in the composition of the proteins, due to which dense scar structures are formed. The use of pectin with an esterification degree of not more than 50% allows the formation of ordered egg-box matrix structures into which other components of the material can be involved and reversibly immobilized due to electrostatic interactions. The use of pectin with an esterification degree exceeding 50% does not provide the formation of stable hydrogels.

The introduction of pectin at a concentration below 0.5% does not allow to obtain stable hydrogels suitable for creating an implantable matrix material, and an excess of pectin concentration of more than 2.0% in the composition of the material leads to the formation of a dense matrix with a large modulus of elasticity, which does not provide effective penetration of cells and their processes, necessary for tissue regeneration.

The introduction of collagen of type I in the amount of 0.1-1.5% contributes to the achievement of the claimed technical result, while the use of concentrations of less than 0.1% does not provide the material with the necessary adhesive properties, which does not contribute to the growth of nerve conductors and does not provide effective regeneration . In addition, a decrease in concentration below the specified value does not give the implantable material stability. An excess of type I collagen concentration of more than 1.5% in the composition, as in the case of pectin, leads to the formation of a dense matrix with a large elastic modulus, which does not ensure the effective penetration of cells and their processes, necessary for tissue regeneration.

The use of type IV collagen in the implantable matrix material in final concentrations of 0.01-0.5% ensures the realization of the regenerative potential of cells, brings the composition of the composite matrix closer to that in developing embryonic structures, which are characterized by active cell migration along pathways marked with this type of collagen , which stimulates, in particular, the regeneration of nerve conductors. A decrease in concentration below 0.01% is not an effective incentive for the growth of cell processes, and an excess of concentration of more than 0.5% inhibits their growth.

The use of collagen type IV in the form of a preparation of its NC1 hexamers as part of the implantable matrix material increases the efficiency of the use of type IV collagen as a matrix component that stimulates the migration of cells and the growth of their processes.

The claimed technical result is also achieved by methods of obtaining implantable matrix material.

The inventive implantable matrix material for regenerative medicine obtained by mixing at temperatures not exceeding 5 ^{° C} acetic acid solution of collagen type I to its final concentration in the matrix material of 0.1-1.5% with type IV collagen solution to its final concentration in the matrix in the form of hydrogel material 0.01-0.5%. Then, the resulting mixture is mixed with a pre-prepared gelation initiator, including a solution of calcium chloride in a concentration that ensures the gelation process, a non-toxic buffer system in an amount that ensures neutralization of the mixture, and a sodium chloride solution to a physiological concentration; then a pectin solution with an esterification degree of not higher than 50% to a final concentration of 0.5-2.0% is introduced into the resulting mixture and a gel is formed by raising the temperature of the mixture to physiological.

The sequence of procedures for mixing the components is fundamentally important, since this is precisely what ensures the stated result. Deviation from this scheme, in particular, mixing of type I collagen acetic acid solution directly with pectin solution, causes both components to precipitate, which does not lead to the formation of a matrix material in the form of a hydrogel. And the primary mixing of the pectin solution with the initiator of gelation leads to the rapid formation of the gel and the impossibility of introducing extracellular matrix proteins into its composition, which does not allow to obtain composite material.

Mixing an acetic acid solution of type I collagen to its final concentration in the matrix material 0.1-1.5% with a solution of type IV collagen to its final concentration in the matrix material 0.01-0.1%, followed by the introduction of the previously prepared gel initiator at a temperature above 5 ^{° C} leads to the rapid formation of a protein gel that does not allow to include in its composition the pectin.

The use of calcium chloride in the gel initiator, which is 1-10 mM in the final concentration in the matrix material, provides a gel process and allows the formation of ordered egg-box matrix structures that form a gel matrix by associating pectin molecules with a low degree of esterification. The introduction of a non-toxic buffer system into the gel initiator in an amount that ensures the neutralization of the collagen mixture leads to the formation of gels due to the association of collagen molecules.

As a non-toxic buffer system in the method, in particular, NaOH-N-2-hydroxyethylpiperazine-N′-ethanesulfonic acid (HEPES), NaOH-N-2-hydroxyethylpiperazine-N′-3-propanesulfonic acid (EPPS) can be used.

The introduction of sodium chloride in the initiator is necessary to create physiological conditions that are isotonic with respect to mammalian cells.

The formation of hydrogels is stimulated by an increase in temperature to physiological values characteristic of the mammalian organism.

Lyophilized drying of the formed matrix material in the form of a hydrogel allows one to obtain a porous matrix material in the form of a sponge, which helps to expand its field of application, since such a matrix provides a high rate of population of cells, their accelerated migration into the material, which can be an advantage when creating bio-artificial tissue analogues in laboratory conditions.

The claimed technical result is also achieved by a second method of producing the implantable matrix material for regenerative medicine in the form of hydrogel, which consists in mixing at a temperature of not higher than 5 ^{° C} acetic acid solution of collagen type I to a final concentration of 0.1-1.5% with Type IV collagen solution to final concentration of 0.01-0.1%. Then, sodium chloride is added to the extracellular matrix protein mixture to a physiological concentration and a non-toxic buffer system in an amount to neutralize the mixture. After neutralization, a pectin solution with an esterification degree of not higher than 50% to a final concentration of 0.5-2.0% is added to the mixture and only after that a gel is formed.

Mixing extracellular matrix proteins with sodium chloride to a physiological concentration and with a non-toxic buffer system in an amount that ensures the neutralization of the mixture, then allows you to enter the pectin solution and obtain a liquid composition, which is subsequently formed into a gel matrix in various ways.

In one embodiment, the gel formation is carried out by layering on the surface of the liquid composition a solution of a gelation initiator, including calcium chloride in a concentration that ensures the gelation process, a non-toxic buffer system that maintains a neutral pH and sodium chloride to a physiological concentration in the matrix material, followed by a temperature increase to physiological .

This makes it possible to obtain implantable matrix materials of various geometries, since a previously prepared liquid composition can be placed in various vessels, formed smooth surfaces, and only then perform the gel formation procedure by creating conditions for gel formation. This is important both when conducting research on biocompatible materials, and for creating implants of various shapes and purposes.

In another embodiment, the formation of the gel is carried out by introducing a liquid composition directly into the area of implantation in the recipient's body. The presence of free calcium ions in the body fluids and the temperature close to ³⁷ ° C provide conditions of gel formation directly in the implantation area. This allows you to abandon the dissection of tissues to obtain an open surgical field and choose a less traumatic method of introducing an implantable structure by injection.

Examples of the invention.

To prepare the material using sterile components.

Example 1

A gel initiator is preliminarily prepared by mixing 4 μl of 10M NaOH, 300 μl of 1M HEPES (titrated with sodium hydroxide solution to pH 7.4), 300 μl of 5M NaCl, 60 μl of 1M CaCl ₂ , 2.736 ml of deionized water

To prepare 10 ml of implantable matrix material is mixed under aseptic conditions at ⁰ ° C 1.1 ml type I collagen solution with a concentration of 4.5 mg / ml, prepared in 0.03 M acetic acid; 0.5 ml of type IV collagen solution in the form of NC1-hexamers with a concentration of 1 mg / ml and 3.4 ml of a gelation initiator. Then 5 ml of solution is administered a concentration of 3% of pectin having a degree of esterification of 30%, mixed thoroughly and gel formation is carried out in an oven at 37 ^C. The resulting matrix material is a transparent gel which contains 1.5% pectin having a degree of esterification of 30%; 0.5% collagen type I, 0.05% collagen type IV.

Example 2

A gel initiator is preliminarily prepared by mixing 9 μl of 10M NaOH, 300 μl of 1M HEPES (titrated with sodium hydroxide solution to pH 7.4), 300 μl of 5M NaCl, 100 μl of 1M CaCl ₂ , 1.691 ml of deionized water.

To prepare 10 ml of implantable matrix material is mixed under aseptic conditions at a temperature of 5 ^{° C.} 2.5 ml type I collagen solution with a concentration of 6 mg / ml, prepared in 0.03 M acetic acid; 0.1 ml of a type IV collagen solution with a concentration of 1 mg / ml and 2.4 ml of a gelation initiator. Then administered 5 ml of a 4% pectin having a degree of esterification of 50%, mixed thoroughly and gel formation is carried out in an oven at 37 ^C. The resulting matrix material is a transparent gel, containing 2% pectin having a degree of esterification of 50%, 1.5 % collagen type I, 0.01% collagen type IV.

Example 3

Pre initiator gel was prepared by mixing 4 ul 10M NaOH, 300 .mu.l of 1M EPPS (titrated with sodium hydroxide solution to pH 7.4), 300 l of 5M NaCl, 40 .mu.l of 1M CaCl _2, 1,356 ml of deionized water.

To prepare 10 ml of implantable matrix material is mixed under aseptic conditions at a temperature of 5 ^{° C.} 1 ml of type I collagen solution with a concentration of 1 mg / ml, prepared in 0.03 M acetic acid; 2 ml of type IV collagen solution with a concentration of 2.5 mg / ml and 2.0 ml of a gel initiator. Then 5 ml of solution is administered a concentration of 1% pectin having a degree of esterification of 10%, mixed thoroughly and gel formation is carried out in an oven at 37 ^C. The resulting matrix material is a transparent gel, containing 0.5% of pectin having a degree of esterification of 10% 0 , 1% collagen type I, 0.5% collagen type IV.

Example 4

To prepare 10 ml of implantable matrix material aseptically in a petri dish is mixed at ⁰ ° C 1.1 ml of collagen type I solution with a concentration of 4.5 mg / ml, prepared in 0.03 M acetic acid; 0.5 ml of type IV collagen solution in the form of 1 mg / ml NC1-hexamers. To the resulting mixture were added 300 μl of 1M HEPES (titrated with sodium hydroxide solution to pH 7.4), 4 μl of 10M NaOH, 300 μl of 5M NaCl, 2.796 ml of deionized water. Then 5 ml of a solution of pectin with a concentration of 3% with a degree of esterification of 30% are introduced, mix thoroughly. On the surface, containing 12 mM calcium chloride, the resulting liquid composition was layered gently equal volume of a solution of 150 mM NaCl, 50 mM HEPES (pH 7.4), then subjected to the formation of gel in an oven at 37 ^C. The resulting matrix material is formed into a reservoir gel with smooth smooth surfaces contains 1.5% pectin with an esterification degree of 30%, 0.5% collagen type I, 0.05% collagen type IV.

Example 5

To prepare 10 ml of implantable matrix material is mixed under aseptic conditions at a temperature of 5 ^{° C.} 1 ml of type I collagen solution with a concentration of 1 mg / ml, prepared in 0.03 M acetic acid; 2 ml of type IV collagen solution with a concentration of 2.5 mg / ml and neutralized by adding 300 μl of 1M HEPES (titrated with sodium hydroxide solution to pH 7.4) to the resulting mixture, 4 μl of 10M NaOH, 300 μl of 5M NaCl, 1.396 ml of deionized water . Then, 5 ml of a 3% pectin solution with an esterification degree of 30% is introduced, mixed thoroughly and the syringe filled with the resulting liquid composition. Then the liquid composition is administered to rats by injection into the implantation area. After 1 and 10 days after implantation, the animals were removed from the experiment by decapitation. Tissue samples from the implantation area were examined for the presence of formed matrix material. Studies have shown that both with subcutaneous implantation and in the case of implantation in the area of traumatic damage to the spinal cord, the injected liquid composition formed a matrix material, which is a gel. In the study of histological sections of these samples for a period of 10 days after implantation, fragments of the implanted matrix material containing cellular elements were observed.

Example 6. The matrix material is obtained according to example 2, then carry out its freeze drying. As a result, pores are formed in the formed gel, and the finished product itself is a biopolymer sponge.

To prove the functional performance of the claimed inventions, the following experiments were carried out. The results of the experiments are shown in figures 1-8.

In FIG. 1. presents the results of the cultivation of neural stem cells of rat fetal brain on various substrates, where: FIG. 1A on polystyrene, FIG. 1B — on pectin, FIG. 1B — on a matrix (claimed) material in the form of a hydrogel.

In FIG. 2. presents the morphology of implants based on pectin hydrogel (Fig. 2A and Fig. 2B), the inventive matrix material (Fig. 2B and Fig. 2G), collagen (Figs. 2E and 2E) on slice preparations 10 days after subcutaneous implantation to rats. In FIG. 2, the following notation is used: KG - collagen sponge, KM - composite matrix material, M - macrophages, FC - fibrous capsule, C - vessels.

In FIG. 3. presents the morphology of implants based on pectin (Fig. 3A, Fig. 3B), the inventive matrix material (Fig. 3B, Fig. 3G, Fig. 3D, Fig. 3E) for 1 month after subcutaneous implantation (Fig. 3A, Fig. 3B, Fig. 3G), 3 months after implantation (Fig. 3B, Fig. 3D, Fig. 3E). In FIG. 3 the following notation is used: KM - composite matrix material, M - muscles, FC - fibrous capsule. The double arrow in FIG. 3B denotes the implantation area.

In FIG. 4. presents images of the spinal cord of rats on magnetic resonance imaging 4 days after acute spinal injury and implantation of a collagen sponge (Fig. 4A) and the inventive matrix material (Fig. 4B).

In FIG. 5. images of frame-by-frame video recording of various groups of experimental animals in the process of movement, where: FIG. 5A from the control group, FIG. 5B - from the group "Pectin gel" and Fig. 5B - from the Matrix Material group 60 days after spinal segmentectomy.

In FIG. 6. shows the appearance of the hind limbs of rats from the control group (Fig. 6A), the "Pectin gel" group (Fig. 6B) and the "Matrix material" group (Fig. 6B) on the 60th day after the spinal cord segmentectomy.

In FIG. 7. presents the histological structure of the spinal cord of rats from the experimental group "Matrix material" 3 months after surgery. Stained with hematoxylin and eosin (Fig. 7A, Fig. 7G); immunohistochemical detection of β-III-tubulin, secondary antibodies conjugated to Alexa Fluor 546 (Fig. 7B, Fig. 7B). Images were obtained by the bright field method (Fig. 7A, Fig. 7G), epifluorescence microscopy (Fig. 7B) and confocal laser scanning microscopy (Fig. 7B is a merged image obtained in transmitted light and reflected light). FIG. 7A is a general view; FIG. 7B is a region indicated by a rectangle in FIG. 7A; FIG. 7B - region of the scar, arrows indicate the processes of nerve cells germinating in the gel; FIG. 7G - macrophages in the scar area, where: M - macrophages; NS - neuronal structures.

In FIG. 8. presents the histological structure of the spinal cord of rats from the control group 3 months after surgery. FIG. 8A is a general view, hematoxylin and eosin staining; FIG. 8B, FIG. 8B is a region indicated by a rectangle in FIG. 8A; FIG. 8B - immunohistochemical detection of β-III-tubulin (secondary antibodies conjugated to Alexa 546); FIG. 8B is the staining of DAPI cores. Images were obtained by the bright field method (Fig. 8A), epifluorescence microscopy (Fig. 8B, Fig. 8B). Where: HB - nerve fibers, SC - capsule wall.

To assess the biocompatibility of materials in vitro, rat fetal neural stem cells were cultured using the samples obtained in Example 1 as a matrix substrate. As a control, cells were applied to the bottom of the polystyrene plate wells or incubated on the surface of a gel formed from a pectin solution. The results of culturing neural stem cells are presented in FIG. 1. In FIG. 1A presents the result of culturing cells on polystyrene, where individual cells of a unipolar, bipolar and triangular shape with short processes were found, however, most of the cells retained a spherical shape, and a significant part of the cells formed typical conglomerates of cells - neurospheres. In the culture of neural cells on the surface of the hydrogel formed from the pectin solution (Fig. 1B), scattered process cells were practically not observed, however, as in the previous case, the formation of dense neurospheres with rare seeding of cells on the periphery was noted for this sample; the formation of processes and pronounced signs of differentiation were not observed for cells cultured on this material. In the culture of neural stem cells on the surface of the matrix material in the form of a hydrogel (Fig. 1B), the formation of typical neurospheres was observed, however, the nature of the spreading of cells on the periphery of the neurospheres on the substrate surface and the morphology of the processes were significantly different from those of the control cultures. So, in the culture of neural cells on matrix material, the formation of long processes extending over considerable distances from the boundaries of the neurospheres was observed. Cell viability in all three cultures was not significantly different. This proves that the obtained matrix material is able to maintain viable cell cultures, is not toxic, and ensures the formation of long processes of nerve conductors.

In another experiment, the biodegradation rate of materials was checked. For this purpose, from the samples obtained in example 4, using a special punch, discs with a diameter of 0.5 cm were cut, which were implanted subcutaneously in rats. In the experiment, 36 white Wistar female rats weighing about 250 g were used. Animals were kept under standard vivarium conditions. The animals were anesthetized by intramuscular injection of a mixture of zoetil (2 mg / kg) and xylazine (4 mg / kg). The hair was cut off on the back and the surgical field was treated with a solution of iodine in 70% alcohol. The skin was cut paramedially and pockets were made with blunt dissection. One implant was placed in each pocket. Each animal was implanted with 4 discs. As a control, disks formed from pectin were implanted in one group of animals, and a sterile collagen sponge was implanted in the other group. The skin was sutured with silk. The wound was re-treated with an alcohol solution and tetracycline. Immediately after surgery and in the next three days, a prophylactic dose of an antibiotic was administered subcutaneously (ceftriaxone 40 mg / g).

Animals were removed from the experiment for 10 days, 1 month, 3 months with deep ether anesthesia. The implants were removed, fixed in 4% paraformaldehyde prepared on phosphate buffer for 24 hours. Samples were washed with phosphate buffer and transferred to absolute ethanol through a series of aqueous ethanol solutions. Poured into paraffin according to standard methods. Cross sections were made with a thickness of 5 μm. Hematoxylin-eosin stained for general morphology.

Throughout the duration of the experiment, there was no deterioration in the condition of the animals. Not a single animal has died. In the area of implantation, no macroscopic signs of inflammation were observed - redness, swelling.

10 days after implantation, significant differences were revealed in the morphology of pectin implants and the inventive matrix material. The pectin matrix has largely degraded. Cells penetrated into it, formed a new tissue surrounding the islands of the still non-degraded matrix (Fig. 2A, 2B). The implant was surrounded by a capsule 99 ± 18 μm thick, consisting of fibroblasts and the collagen fibers synthesized by them (Fig. 2A). Numerous vessels were detected in this layer of fibroblasts. They were the source of macrophages that populated the implant (Fig. 2B).

The developed matrix material degraded to a much lesser extent 10 days after implantation (Fig. 2B). In the field of implantation, a significant part of the implanted material was preserved. The central part of the implant, not populated by cells, was clearly distinguished. Active implant infiltration by cells, primarily fibroblasts, occurred on the periphery. The cells did not populate the implant randomly, but penetrated into it one after another, forming chains of cells parallel to the edge of the implant. The implant was surrounded by a loose fibrous capsule 104 ± 10 μm thick. Active vascularization was observed not only of the connective tissue surrounding the implant, but also of the matrix itself (Fig. 2G). 10 days after implantation, the process of collagen implant infiltration by neutrophils, macrophages and fibroblasts was observed. Moreover, this process was uneven. In some cases (28%), in the early period after implantation, the collagen implant was abundantly infiltrated by cells (Fig. 2D), in other cases (72%), no significant infiltration of the material by cells was observed, a fragment of the collagen implant was surrounded by a dense fibrous capsule 48 ± 12 thick μm (Fig. 2E). After 1 month, fragments of the collagen implant were no longer detected in the area of implantation.

In the area of implantation of pectin and developed matrix material, the cells responsible for the inflammatory response were few. Single lymphocytes were detected, neutrophils were not detected. Obviously, the inflammatory reaction to the implantation process itself has already been completed by this time. The implanted matrices were immunologically inert and did not cause a significant inflammatory reaction.

1 month after implantation, the pectin matrix was substantially resorbed and replaced by the recipient's own tissues. At this time, the transformation of tissue formed in the implantation area was observed. The fabric acquired a mesh look (Fig. FOR). The cells of this grid merged in places in cavities lined with spindle-shaped narrow cells - fibrocytes. Inside the cavities there were process cells with a slightly stained cytoplasm, a round or oval nucleus - fibroblasts. The fibrous capsule decreased, its thickness was 32 ± 12 μm. After 3 months, the remodeled implantation zone decreased in size, while maintaining the same morphology as for 1 month (Fig. 3B). The fibrous capsule surrounding the implant has completely disappeared.

In the developed matrix, 1 month after implantation, the number of cells increased, they uniformly populated the matrix (Fig. 3B, 3G). The matrix material slowly degraded, after 3 months its quantity in the implantation area decreased, but still remained significant. The fibroblasts that populated the matrix remodeled the implanted material, changing its morphology, as a result of which the tissue formed at the site of the implant began to acquire a mesh appearance (Fig. 3D). In FIG. 3E shows the morphology of the implantation area after 3 months at a higher magnification, on which fragments of matrix material surrounded by cells are clearly distinguishable. The fibrous capsule surrounding the implant has decreased. Its thickness was 47 ± 22 μm.

The collagen sponge implanted subcutaneously was resorbed within 2-4 weeks. After 1 month, fragments of the collagen sponge were no longer detected in the area of implantation. 10 days after implantation, the process of collagen sponge infiltration by neutrophils, macrophages and fibroblasts was observed. Moreover, this process was uneven. In some cases (28%), in the early period after implantation (10 days), the sponge was abundantly infiltrated by cells (Fig. 2B), in other cases (72%) there was practically no cell infiltration of the material, and a fragment of the collagen sponge was surrounded by a dense fibrous capsule 48 ± 12 μm thick (Fig. 2D).

From the presented results it follows that the inventive matrix material obtained by the developed methods has a low biodegradation rate in the mammalian organism, is biocompatible and non-toxic in vivo.

For a final assessment of the health of the claimed inventions, an experiment was conducted in which samples of the matrix material obtained in Example 1 were implanted in the area of traumatic damage to the spinal cord of rats. As a control, one group of animals was implanted with material formed from pectin, and a sterile collagen sponge was implanted in the other group.

A model of acute spinal cord injury was created by removing a 2 mm long fragment of the spinal cord at the level of 12 thoracic - 1 lumbar vertebra, according to Werley S., Pinet E., De Robertis L. et al. Heterogeneous PHPMA hydrogels for tissue repair and axonal regeneration in the injured spinal cord // Journal of Biomaterials Science, Polymer Edition. 1998. Vol. 9. P. 681-711; Woerly S., Doan D., Sosa N. et al. Reconstruction of the transected cat spinal cord follow -ing NeuroGel ™ implantation: axonal tracing, immunohistochemical and ultrastructural studies // International Jornal of Developmental Neuroscience. 2001. Vol.19. P. 63-83).

Anesthesia was performed by inhalation of medical ether. The hair on the back of the animal was treated with an antiseptic solution. They shaved the surgical field. The area of the surgical field was treated with 70% ethyl alcohol.

The skin was dissected along the back, longitudinally, above the spine from 9 thoracic to 3 lumbar vertebrae. The subcutaneous fascia was separated with scissors at the level of the skin incision. On two sides of the spinous process of the 12 thoracic vertebra (T12), close to it, 2 parallel incisions were made, approximately 15 mm long, separating the muscles from the surface of the vertebra.

A laminectomy was performed at the level of the 12 thoracic vertebra, providing direct access to the spinal cord. The spinal cord was crossed with a disposable scalpel blade. Bleeding was stopped by a hemostatic collagen sponge (OAO Luga Plant BELKOZIN). After stopping the bleeding, a fragment of the spinal cord was cut off with a length of approximately 2 mm.

After complete hemostasis, the resulting spinal cord defect in the first control group was filled with a collagen sponge. In the second control group, the defect was filled with pectin gel, in the experimental group - with the claimed matrix material. The implant was isolated with a collagen membrane. After that, the wound was sutured in layers.

Within three days after the operation, a course of antibacterial therapy was carried out: 100 μl of 5% ceftrioxone intraperitoneally 2 times a day. Inspection of the operated animals was carried out daily for 3 months until the end of the experiment. The urine was lowered twice a day. Trophic disorders were assessed by the general appearance of rats, weight loss, hairline, trophic ulcers, pressure sores.

Animals were removed from the experiment for 10 days, 1 month, 3 months with deep ether anesthesia.

Of all the rats operated, 17 animals made up the first control group - Collagen Sponge. The second control group - the group "Pectin gel" - included 7 animals. The experimental group - the matrix material group - included 19 animals.

The results of implantation of three types of biopolymer matrices into the spinal cord defect region of rats revealed significant differences between the studied groups.

Magnetic resonance imaging tomography of rat spinal cord on day 4 after surgery showed that in the first control group, the defect in which was filled with a collagen sponge, the area of injury was characterized by severe edema (Fig. 4A). The effect of the development of edema in the "Matrix material" group was expressed significantly less (Fig. 4B).

Analysis of the motor activity of animals was carried out on the basis of video recordings, three frames of which for each group are presented in FIG. 5. In the first control group, half of the animals (55.5%) retained hind limb paraplegia during the entire observation period. Starting at 14 days after the operation, only weak movements in the hip joints were recorded, further improvement did not occur (Fig. 5A). The rest of the animals had a restoration of their motor functions, which reached a maximum of 8 weeks after surgery and averaged 4.3 points on the BBB locomotor activity rating scale. In 42.8% of the animals hypertension of one of the limbs was observed. Autotomy phenomena were observed in more than half of the animals — the animals gnawed off their hind limbs or tail, as shown in FIG. 6, which shows the appearance of the hind limbs of rats from the first control group (A), the second control group - “Pectin gel” (B) and the group “Matrix material” (C) on the 60th day after the spinal cord segmentectomy. For animals from the Matrix Material group, a normal view of the hind limbs was noted (Fig. 6B).

In the group "Pectin gel" did not observe the restoration of motor activity of experimental animals (Fig. 5B). Only weak movements in the hip joints were observed in animals (1-2 points on the BBB scale). A distinctive feature of this group was the absence of pronounced phenomena of autotomy and hypertonicity (Fig. 6B).

In the "Matrix Material" group, a rather strong scatter of data was observed for such indicators as restoration of motor activity, autotomy, and hypertonicity. In 1 rat, there was no restoration of motor activity until the end of the experiment, but 1 hind limb was moving immediately after the operation. Perhaps the presence of one working hind limb impeded the physiological recovery of the second limb. Another 2 rats also did not have recovery, but they were taken out of the experiment for a period of 10 days, so their further fate is not known. Although in general in other animals of this group at this time, motor activity is estimated from 1 to 6 points - from movements in the hip joints to active movements in the hip, knee and ankle joints.

Three months after the operation, rats from the experimental group “Matrix material” noted a further improvement in the motor activity of the hind limbs. In this group, rats capable of independent movement using both hind limbs and their periodic coordination with the front were observed. In one rat, motor activity was restored in only one hind limb. In some rats, both hind limbs maintained their physiological position and were used for walking, although the weight was not maintained by the plantar surfaces of the paws. In general, the restoration of the motor activity of the hind limbs of rats from the “Matrix material” experimental group ranged from 5 to 15 points on the “BBB” scale three months after implantation. The maximum recovery of motor activity of animals in 15 points corresponded to stable plantar walking and coordination between the front and rear limbs (Fig. 5B).

Histological analysis of the spinal cord of rats subjected to segmentectomy and subsequent reconstructive therapy revealed the following features in the groups.

The histological preparations of the spinal cord of rats from the groups “Collagen Sponge” and “Matrix Material” that were withdrawn from the experiment in the early postoperative period (10 days after segmentectomy and reconstructive therapy) were analyzed.

In the Collagen Sponge group, in the area of spinal cord injury, an experimentally created cavity filled with a collagen sponge was observed. Inside the sponge, in some places, accumulations of red blood cells and lymphocytes were noted. No massive infiltration of the implant by the recipient cells was observed. The sponge did not contribute to the consolidation of the rostral and caudal fragments of the brain, separated by trauma.

In the Matrix Material group, at the same time, the restoration of the physical integrity of the spinal cord through consolidation of the rostral and caudal fragments using the material was noted. The part of the scar adjacent to the rostral part of the brain is represented by a homogeneous loose connective tissue scar, in which the remains of the gel are almost not detected.

The central region of the scar is filled with numerous fragments of the gel, separated by strands of tissue, in which new vessels are also revealed. During immunocytochemical labeling of neuronal structures with antibodies against β-III-tubulin, the rostral and caudal parts of the brain are intensively stained. In the area of the scar, single nerve fibers are detected.

In the gray matter of the spinal cord in the areas adjacent to the area of injury, neuron death is observed, especially pronounced in the caudal part in the control group. In the “Matrix material” group, despite the destructive processes, the gray matter in the caudal part is in a more preserved form.

The histological preparations of the spinal cord of rats from the Matrix Material group were analyzed 1 month after surgery. A morphological picture of the structure of the tissues of the rumen and adjacent tissues of the spinal cord was observed, which is different from the control group. In animals of this group, a friable scar was formed in which neuronal structures marked with antibodies against β-III-tubulin were detected. The astrocytic reaction at the border of the rostral intact part of the brain and the scar was weak, there was no dense network of astrocytes and their processes, which usually plays an important role in blocking regenerating axons. Astrocytes in the rumen were not detected.

3 months after the operation in the Matrix Material group, the most typical picture of the structure of the spinal cord of experimental animals was as follows. The expressed consolidation of the rostral and caudal fragments was revealed (Fig. 7). In the area of the injury, numerous fragments of the gel were still detected, separated by strands of tissue in which neuronal structures were detected by immunohistochemistry. In FIG. 7A shows a histological picture in the area of traumatic injury and implantation, revealed by routine general morphological staining of sections with hematoxylin and eosin. FIG. 7B shows the result of detecting neuronal structures by indirect immunohistochemical labeling with anti-β-III-tubulin antibodies and imaging using epifluorescence microscopy. The area of the scar was studied in detail by confocal laser microscopy, which confirmed the presence of a significant number of neuronal structures (NS), germinating in the implant (Fig. 7B). In FIG. 7B shows that the processes of neurons penetrated directly into the gel, which confirms that this matrix material supports the regeneration of nerve conductors. Macrophages were visible in the scar region, indicating an ongoing implant biodegradation process (Fig. 7G).

In the control group, the structure of the spinal cord for a period of 3 months after surgery was also heterogeneous among various animals. One of the common options - obviously the most unfavorable - was the formation of a post-traumatic capsule in the area of injury, which prevented the regeneration of nerve fibers (Fig. 8A). A certain number of nerve fibers sprouted along the periphery, circling the capsule, but they were not able to effectively overcome this barrier (Fig. 8B). The capsule consisted of a significant number of cells, as evidenced by the staining of histological preparations using DAPI (Fig. 8B). The internal contents of the capsule are also represented in part by cells and non-cellular contents. In another case, polymorphonuclear neutrophils were distinctly detected in the same capsule. Probably, the capsule was formed as a result of too strong an inflammatory process and massive leukocyte infiltration.

In other cases, the formation of an overly dense scar was observed in animals. At the border of the spinal cord and the scar, a degenerative process continues, accompanied by the formation of pathological cavities. Numerous macrophages are observed in this area. No fragments of the implanted matrix were detected at this time, and the scar tissues themselves are very tightly packed collagen fibers oriented in the transverse direction, which was revealed by a specific color using red picrosirius. Obviously, axons were not able to overcome such a barrier. The density of astrocytes at the border of the preserved tissue of the spinal cord and scar is increased, compared with normal tissue of the spinal cord, as well as with the area of implantation of the matrix gel in the experimental group described above. In the distal part of the scar, nerve cells and their processes are not detected. In the control group there were also animals that showed some restoration of motor activity of the hind limbs, which was generally lower than in the "Matrix material" group.

As a result of the study, it was possible to establish that the claimed inventions make it possible to create an implantable matrix material with not only biocompatibility demonstrated in experiments in vitro and in vivo with subcutaneous implantation, but also providing regeneration of the damaged brain, stimulating partial restoration of nerve conductors and preventing the formation of dense structures of the scar, which is an insurmountable barrier in neuroregeneration.

The above results confirm that a composite matrix material has been created for the needs of regenerative medicine and tissue bioengineering, which has the following advantages.

1. The material is biocompatible and weakly immunogenic, which distinguishes it most favorably from the known materials based on chitin and chitosan, which are characterized by fairly pronounced immune reactions despite a number of similar physicochemical and functional properties.

2. The material is able to efficiently provide tissue regeneration processes, in particular when implanting laboratory animals (rats) into the area of traumatic damage to the spinal cord of the laboratory animals (rats), it helps to restore locomotor functions of rats to 8.5 ± 3.3 points on the BBB scale, which is significantly higher than using all of these analogues.

3. The material has the property of slow biodegradation, provided by the combination of a modified plant polysaccharide (pectin with an esterification degree of up to 50%) and extracellular matrix proteins in the composition of the composite matrix, which contributes to the preservation of the implanted material in the body of mammals for several months. This rate of biodegradation is commensurate with the slow regeneration of brain tissue and therefore provides an effective repair process. In this regard, the material has an advantage over those based on collagen, as well as hyaluronic acid and its derivatives, which are degraded in the body of mammals within 1 month, while the repair process requires a period of several months.

4. The developed matrix has the ability to limit the formation of dense structures of the scar and cysts with a dense membrane that occur during the natural repair process after traumatic brain damage due to migration and adhesion of neuroglia and connective tissue cells. Although the structures of the forming scar contribute to the restoration of brain integrity, they nevertheless form an insurmountable barrier to the growth and regeneration of nerve cell processes. This property, characteristic of the developed matrix, is an advantage over pure protein matrices (based solely on collagen, laminin, fibronectin), which, due to the very high adhesive properties for neuroglia and connective tissue cells, cause the formation of a denser scar structure and can create obstacles to axon regeneration.

5. The developed matrix material and methods for its preparation provide such a scheme for the formation of material from individual components, in which the process of formation of hydrogel structures requires exclusively physiological environmental conditions and additional components initiating gelation, which are not toxic and are used in physiological concentrations. The process of forming a material in the form of a composite hydrogel proceeds at 37 ° C, which makes possible the final formation of the material inside the recipient’s body, which thus allows the specialist to implant not only preformed products based on the composite material (obtained by preforming), but also to use the injection form of administration postforming material within the recipient’s body. This can be convenient when reconstructing minor tissue injuries or in the absence of an open surgical field available for surgical procedures.

6. Compared with most analogues, the material has high immobilization properties in relation to biogenic and xenogenic substances of various chemical structures. Due to the use of pectins with a low degree of esterification, ordered cellular structures of the “egg-box” type (“egg boxes”) are formed during gelation. Such structures have the property of attracting various substances into the cellular structure. This property of the pectin component allows you to effectively immobilize other components of the material.

Claims (10)

1. An implantable matrix material for regenerative medicine, including a biocompatible polymer mixture of plant polysaccharide and extracellular matrix proteins of type I and IV collagen, characterized in that pectin with an esterification degree of not more than 50% is used as plant polysaccharide, with final component concentrations in the matrix material are in the range of weight. %:
pectin with an esterification degree of not more than 50% 0.5-2.0 type I collagen 0.1-1.5 type IV collagen 0.01-0.5 2. The implantable matrix material according to claim 1, characterized in that a preparation of its NCl hexamers is used as type IV collagen. 3. A method of obtaining an implantable matrix material for regenerative medicine in the form of a hydrogel, which consists in mixing at a temperature not higher than 5 ° C of an acetic acid solution of type I collagen to a final concentration of 0.1-1.5 wt.% With a type IV collagen solution to a final concentration 0.01-0.1 wt.%, Then the resulting mixture is mixed with a pre-prepared gelation initiator, including a solution of calcium chloride in a concentration that ensures the gelation process, a non-toxic buffer system in an amount, providing neutralizing the mixture, and a solution of sodium chloride to physiological concentration; after which a pectin solution is introduced with an esterification degree of not higher than 50% to a final concentration of 0.5-2.0 wt.% and a gel is formed by raising the temperature of the mixture to physiological. 4. The method according to p. 3, characterized in that as a type IV collagen use a preparation of its NCl-hexamers. 5. The method according to p. 3, characterized in that the formed gel is subjected to freeze drying. 6. A process for preparing an implantable matrix material for regenerative medicine in the form of a hydrogel comprising mixing at a temperature of not higher than 5 ^{° C} acetic acid solution of collagen type I to a final concentration of 0.1-1.5 wt.% Of the type IV collagen solution to a final concentration 0.01-0.1 wt.%, Then the resulting mixture is neutralized, stabilized with a non-toxic buffer system and sodium chloride is added to a physiological concentration, after which a pectin solution with an esterification degree of not higher than 50% is added to a final concentration and 0.5 to 2.0 wt.%, obtaining a liquid composition from which the gel is formed. 7. The method according to p. 6, characterized in that as a type IV collagen use the preparation of its NC1-hexamers. 8. The method according to p. 6, characterized in that the gel is formed by layering on the surface of the liquid composition a solution of a gelation initiator, including calcium chloride in a concentration that ensures the gelation process, a non-toxic buffer system that maintains a neutral pH and sodium chloride to a physiological concentration in the matrix material, followed by a rise in temperature to physiological. 9. The method according to p. 8, characterized in that the formed gel is subjected to freeze drying. 10. The method according to p. 6, characterized in that the formation of the gel is carried out by introducing a liquid composition directly into the area of implantation in the body of the recipient.

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