RU2521194C2 - Matrix for cell transplantology - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to cell transplantology and tissue engineering, and describes a matrix, a basic element of which is a flat plate made from a spatially cross-linked hydrophobic polymer containing hydrophilic groups and forming on the plate surface a layer of saturated hydrocarbons having a chain length of 8 to 16 carbon atoms and directed preferentially along the normal line to the plate surface. The spatially cross-linked polymer is formed on substrates by exposing a photopolimerisable composition containing oligourethane methacrylate, a methacrylate monomer having a chain length of 8 to 18 carbon atoms, 2,2-dimethoxy-2-phenylacetophenone and 2,4-ditertbutylorthquinone, to light at a wave length of 320-380 nm.

EFFECT: matrix has a good adhesive capacity; it is biocompatible and bioresorbable, preserves progenitor cells, promotes their differentiation and growth, and is mechanically strong.

5 cl, 1 tbl, 5 ex, 8 dwg

Description

The invention relates to materials used in cell transplantology and tissue engineering to restore the lost structure or function of organs and tissues. In particular, the present invention is capable of being an implant that can be used to replace the internal tissues of any part of the soft organs, to heal a wound, to regenerate tissue and to restore an organ in general, especially in the developing and adult nervous system or in other similar areas of therapy.

Many different types of matrices are known that are attempted to be used in cell transplantology. Two groups can be distinguished among them: (1) using fragments of biological tissues and (2) synthetic materials.

Group (1) includes materials on patents RU 2265446 “IMPLANT FOR THE TREATMENT OF PARKINSON'S DISEASE”, RU 2216281 “METHOD FOR PLASTIC DEFECT OF THE SPINAL BRAIN WITH VEHICLES WITH NON-TERRESTRIC TREATMENT”. The main disadvantage of all matrices using fragments of biological tissues is that they cause an undesirable nonspecific protective reaction of macrophages, and allo and xenografts have an immunological and infectious risk. A common drawback of all types of natural matrices is unpredictable (uncontrolled) resorption, which significantly slows down the formation of new tissue.

To the group (2) include materials Patent RU 2375080, "A method of stimulating nerve regeneration BASED USE BIOCOMPATIBLE suspensions or slurry SILICON NANOCLUSTERS», RU 2394593 "Implantable NEYROENDOPROTEZNAYA SYSTEM, METHOD FOR ITS PREPARATION AND METHOD OF reconstructive neurosurgical operations», RU 2198686 " "POLYMER HYDROGEL OF ACRYLAMIDE COPOLYMER OF THERAPEUTIC USE AND METHOD FOR ITS PRODUCTION". The main disadvantage of using suspensions of silicon nanoclusters is that they do not contribute to the functional regeneration of the conduction nerve pathways. In this case, silicon nanoclusters themselves act in the tissue of the central nervous system through artificially created electrical synapses. Electrical synapses are much less characteristic of the nervous system of adult mammals than chemical ones. Excitation at the electrical synapse can occur in both directions, as opposed to chemical synapses, and has a very low degree of control. Connected by electrical synapses, pre- and postsynaptic cells do not have the property of plasticity, which significantly reduces the efficiency of the brain, due to the impossibility of learning and the formation of long-term memory. In addition, the resulting structures from nanoclusters are not surrounded by an electrically insulating layer, unlike individual nerve fibers, which can lead to a process of nonspecific transfer of excitation and simultaneous activation of neurons that are opposite in function. The formation of silicon nanoclusters is a non-specific process, that is, a cross-connection is possible that does not correspond to the physiological pathways of the nerve impulse.

The disadvantage of using polymer hydrogels is their poor biocompatibility. Polymer hydrogels are not a nutrient medium for the cells placed in them and do not have biocompatibility; these substances turn into a scar element that prevents axon growth and graft engraftment. The disadvantage of using a neuroendoprosthetic system is that the use of an artificial prosthesis of an artery to form a conduit limits the penetration of nutrients to cells placed in the endoprosthesis.

The invention, taken as a prototype (patent RU 2198686), relates to a polymer hydrogel for therapeutic applications, acts as a space-filling material and as a frame, which stimulates tissue regeneration, morphogenesis and remodeling in an integrated organ structure. The obtained non-degradable synthetic matrix of a polymer hydrogel with an anisotropic porous structure, with an active area, good adhesion and tissue compatibility is intended for implantation in the structure of soft tissue, especially in the nervous system, which gradually becomes part of the organ.

The material is a copolymer of (a) N-substituted methacrylamide or acrylamide, (b) a crosslinking agent, and (c) a polymerizable material selected from the group consisting of sugar, sugar derivatives, a tissue connecting peptide, tissue proteins with differentiated molecules such as bone-morphogenetic proteins, and a conjugated polymer with antibodies against lipid derivatives, which has elastic deformation ability and has an equilibrium water content of approximately 80%.

A hydrogel is a covalently bonded, opaque, heterogeneous material that preferably has a light-phase separated structure formed by polymer particles of about 1 to 10 microns in size, preferably from 3 to 5 microns, so as to provide a region with relatively large porosity (macropores) where the hydrogel tends to come into contact with the host tissue, and with a relatively fine porosity (mesopores), where it tends to come into contact with the growing tissue. Also, cells or genetically modified cells can be introduced into the polymer network. This occurs by gel trapping at temperatures below zero, that is, by cryogenic polymerization, during which a porous matrix is obtained by which cells are immobilized and by which cells can be reorganized, can grow and / or differentiate for subsequent transplantation. The polymer mixture can be mixed with living cells, while combining the physical properties of the polymer matrix, taking into account the behavior of a typical hydrogel (porosity, stability, guiding surfaces, permeability) and taking into account biological factors of cells (for example, growth factor). It is also possible to use a three-dimensional culture system, which can be used to obtain a culture of various cells in vitro for a long period of time.

The disadvantages of this polymer is that the method of its production leads to the formation of free radicals, which provokes the appearance of undesirable toxic properties. Also, due to the lack of maintaining the shape of the material, its transportation is impossible without violating the formed intercellular connections.

In addition, the gel capture process of nerve cells, including cryo-freezing, can lead to disruption of the processes of functional regeneration. Cell cultures obtained from mammalian embryonic and postnatal brain are not resistant to cryotherapy. In the process of cryopreservation, most of the neurons die, and after removal from cryopreservation, their place is occupied by actively dividing elements - glial cells. The use of cryoprotective agents, such as DMSO (dimethyl sulfoxide), leads to partial destruction of cell membranes, and since the metabolism of neurons differs from the metabolism of constantly dividing cells, membrane restoration does not occur, which further increases the number of neurons killed during cryopreservation.

The aim of the invention is to create a matrix for cell transplantology, which is a synthetic complex with the properties of a biocompatible matrix to create a tissue engineering construct, namely: the absence of cytotoxicity, the maintenance of adhesion, fixation, proliferation and differentiation of cells placed on its surface, the absence of the effect of maintaining inflammation, in including immune, sufficient mechanical strength for the intended purpose and possible bioresorbability by conventional biological of ways, for example by enzymatic hydrolysis or.

The technical result is achieved by creating a matrix for cell transplantology, in which the main element is a flat perforated plate made of a spatially cross-linked hydrophobic polymer containing hydrophilic groups and forming a layer of saturated hydrocarbons on the surface of the plate with a chain length of 8 to 18 carbon atoms oriented mainly along the normal to the surface of the plate. This spatially cross-linked polymer is formed on substrates with a hydrophobic surface by exposure to light with a wavelength of 320-380 nm photopolymerizable composition containing the ingredients:

Oligourethane methacrylate of the following structure:

where n is of the order of 35.

One of the monomers of the methacrylic series with a chain length of 8 to 18 carbon atoms of the following structure:

CH ₃ - (CH ₂ ) _k —O — C (O) —C (CH ₃ ) = CH ₂ , where k is from 7 to 17.

2,2-Dimethoxy-2-phenylacetophenone of the following structure:

2,4-Ditretbutylorthoquinone of the following structure:

while the above components are taken in the following ratio, wt.%:

Oligourethane methacrylate - 15-60,

The monomer of the methacrylic series with a chain length of 8 to 18 carbon atoms is 39-84,

2,2-Dimethoxy-2-phenylacetophenone - 0.4-3.0,

2,4-Ditretbutylorthoquinone - 0.01-0.06.

A distinctive feature of this method of obtaining a matrix for cell transplantology is that the formation process occurs according to a one-stage scheme that excludes any mechanical effects. Any mechanical effect on the polymer is known to provoke the formation of free radicals, which subsequently lead to polymer degradation and undesirable toxic reactions. The specified material has increased stability in biologically active environments, increased resistance to oxidative processes and processes of protein adsorption on the surface, preventing the formation of a coarse connective tissue capsule. This matrix is designed to grow tissue cells, including neuronal cells. First, a matrix is made, and then specified groups of cells are cultivated on it. The process of growing cell cultures consists in the fact that dissociated cells with the required amount of culture medium are planted on this matrix. A distinctive feature of the invention is that a layer of saturated hydrocarbons with a chain length of 8 to 18 carbon atoms, oriented mainly along the normal to the plate surface, is formed on the matrix surface. The hydrophobic ends are facing the nutrient medium and absorb the lipids contained in the nutrient medium, so that the hydrophobic ends of the lipids are facing the hydrophobic ends of the matrix, and the hydrophilic ones are “outward”. The hydrophilic ends of lipids, in turn, absorb proteins contained in the nutrient medium. Thus, a surface similar to the surface of a cell membrane is reproduced. It is such a solution that allows for high adhesion of cultured cells to the matrix, which provides a high density of cell attachment to this substrate. The perforated structure of the main element of the matrix - the plate - allows you to form bonds between groups of cells on two sides of the plate and promotes their interaction. Also, the matrix for cell transplantology can be in the form of a volumetric element consisting of a multilayer complex of perforated plates. Further transplantation of these structures can be used to replace damaged tissue, plastic defect, as well as stimulate the proliferation and differentiation of their own tissues, which may lead to reconstructive treatment of various defects of the nervous tissue after an injury or surgery.

On the claimed matrix were cultured cells of the cortex of the cerebral hemispheres of mouse embryos. During the work, dissociated neuronal cells of the cerebral cortex (CBP) of 18-day-old mouse embryos were cultured in the following stages:

Stage 1. An extraction of the CBP culture of the embryo was performed.

2 stage. The cortex culture was mechanically ground with a scalpel, and the tissue was placed in a 0.025% trypsin solution.

3 stage. The tissue was washed twice with PBS solution, NBM culture medium.

4th stage. The tissue was centrifuged and resuspended in the required amount of culture medium.

5 stage. The suspension was digested in 50 μl for all types of polymers, kept in this form for 2 hours, after which it was filled with NBM1 nutrient medium with the addition of 5% fetal calf serum.

6 stage. The vital activity of the culture was maintained under conditions of a CO ₂ incubator at a temperature of 35.5 ° C and a gas mixture containing 5% CO ₂ .

7th stage. The medium was changed on the first day of cultivation, then 1 time in 3 days.

Cell dissociation was achieved by treating the CBP tissue with 0.25% trypsin (Invitrogen 25200-056). Cells were resuspended in Neurobasal medium Neurobasal ^TM (Invitrogen 21103-049) in conjunction with bioactive B27 supplement (Invitrogen 17504-044), glutamine (Invitrogen 25030-024), fetal bovine serum (PanEco K055). The culture was maintained under conditions of a CO ₂ incubator at a temperature of 35.5 ° C and a gas mixture with 5% CO ₂ in an incubator MSO-18A1C (Sanyo). The structure and dynamics of the development of cultures was evaluated using an inverted microscope DM 1000 (Leica). The composition of neuroglial networks in cultures and the dynamics of spontaneous calcium oscillations reflecting the state of calcium homeostasis of cells forming an in vitro neuroglial network were investigated using a Zeiss LSM510 NLO Duoscan confocal laser microscope. Specific calcium dye Oregon Green 488 BAPTA-1 and Ca ²⁺ -sensitive dye Sulforhodamine 101, selectively labeling glial cells, were used as fluorescent probes. Specific antibodies to surface and intracellular proteins were used to label cells in dissociated cultures. A characteristic marker of glial cells was glial fibrillar acid protein (GFAP), and one of the most actively used markers of neurons was a marker of mature neuron nuclei (NeuN). To visualize the results obtained, the addition of secondary antibodies with a fluorescent label is necessary. We used as secondary antibodies: anti-mouse antibodies labeled with fluorescent dye Alexa-fluo 430 to visualize NeuN protein (Alexa-fluo 430 anti mouse Invitrogen A 11063), and anti-chicken antibodies labeled with fluorescent dye Su-5 to visualize GFAP protein (Cy-5 anti cnk Millipor AP194S).

In the analysis of the dynamics of growth and development of cell culture using an inverted microscope, it was revealed that at the end of the first day of cultivation, viable cells are attached in large quantities to the claimed matrix. On the second day of cultivation, part of the cells is separated from the polymer, the remaining progenitor cells form neurosphere-like structures with processes extending from the center. In the process of division of progenitor cells and the development of strands of glial cells along which neurons are located, new neurosphere-like structures are formed. By the ninth day of cultivation, the development of the glial network around the neurospheres is observed. Further formation on the surface of the substrate of complex plexuses of growing processes of nerve and glial cells is observed, intensive processes of growth, differentiation and proliferation of dissociated cells of the nervous system, and the connection of neurosphere-like structures between themselves by numerous processes are underway. If we talk about the duration of cultivation, then more than 80 days have passed since the first planting and the cultures remain viable, which indicates the absence of toxic properties of this polymer on the substrate. By the method of functional neuroimaging characterizing metabolic changes in cells, spontaneous calcium oscillations were revealed, which manifest themselves in a change in the fluorescence intensity of Oregon Green 488 BAPTA-1, which indicate an active functional state of the neuroglial network of cultured cells. The immunohistochemical method using specific antibodies proved the presence of a differentiated neuronal network cultured on this polymer. It was shown that the neurosphere-like structure is an accumulation of neurons and glial cells that rise above the polymer surface by an average of 200 μm with numerous strands and a developed glial network at its base.

The dynamics of growth and development of cell culture is illustrated by the following figures:

Figure 1. The first day of cultivation - viable cells (1) attach to the matrix in isolation or in small groups.

Figure 2. Perforated matrix structure for cell transplantology.

Figure 3. The second day of cultivation - the remaining progenitor cells form neurosphere-like structures (2) with processes extending from the center (3).

Figure 4. The formation of new neurosphere-like structures (A, B).

Figure 5. By the tenth day of cultivation, the development of the glial network around neurosphere-like structures is observed:

C is a neurosphere-like structure,

D is the glial network.

6. Connection of neurosphere-like structures among themselves by numerous processes of growing neurons and glial cells (twenty-seventh day of cultivation). The figure shows that the matrix for cell transplantology has a perforated structure.

7. - Twenty-fifth day of cultivation.

Fig - Fifty-ninth day of cultivation. A denser and more developed monolayer neuronal and glial network is formed on the matrix for cell transplantalogy.

It was proved that the claimed matrix has good adhesive ability, preserves progenitor cells, promotes their differentiation, stimulates growth, which serves as the basis for the further introduction of this material in clinical practice.

The invention is illustrated by the following examples.

Example 1

The main element of the matrix for cell transplantology - the plate - is made as follows. In a reaction flask equipped with a stirrer, the components are sequentially introduced in the following ratio,% wt .:

Oligourethane methacrylate - 49.55,

Octyl methacrylate (k = 7) - 49.55,

2,2-Dimethoxy-2-phenylacetophenone - 0.89,

2,4-Ditretbutylorthoquinone - 0.01.

The resulting mixture was stirred at room temperature for 40 minutes until completely dissolved. After mixing, the composition is filtered off and pumped out using a vacuum pump at a pressure of 0.5-1 mm Hg. until gas evolution ceases. The composition is poured onto a hydrophobic substrate with a gasket installed along the edge forming the thickness of the plate. Then they cover it with a photo mask containing a transparent pattern on a background opaque to UV light (perforation holes correspond to an opaque background, the rest is transparent), and tightly compress. The resulting design is irradiated with UV light with a wavelength of 320-380 nm from the side of the photomask for a time optimal for reproducing a given geometry. After irradiation, the photomask and the substrate are disconnected. The resulting polymer plate is washed in a suitable solvent from the residues of the uncured composition and dried until the solvent is completely removed. Thanks to the photochemical technology used according to the EPR data, free radicals are absent, the material is stable in biologically active media.

Example 2

The composition is prepared, as in example 1, with the following ratio of components:

Oligourethane methacrylate - 32.27,

Decyl methacrylate (k = 9) - 66.0,

2,2-Dimethoxy-2-phenylacetophenone - 1.7,

2,4-Ditretbutylorthoquinone - 0.03.

A matrix is prepared for cell transplantology, as in Example 1.

Characteristics are given in table 1.

Example 3

The composition is prepared, as in example 1, with the following ratio of components:

Oligourethane methacrylate - 58.0,

Octadecylmethacrylate (k = 17) - 40.5,

2,2-Dimethoxy-2-phenylacetophenone -1.48,

2,4-Ditretbutylorthoquinone - 0.02.

A matrix is prepared for cell transplantology, as in Example 1.

Characteristics are given in table 1.

Example 4

The composition is prepared, as in example 1, with the following ratio of components:

Oligourethane methacrylate - 9.4,

Hexadecylmethacrylate (k = 15) - 90.576,

2,2-Dimethoxy-2-phenylacetophenone - 0.02,

2,4-Ditretbutylorthoquinone - 0.0004.

A matrix is prepared for cell transplantology, as in Example 1.

Characteristics are given in table 1.

Example 5

The composition is prepared, as in example 1, with the following ratio of components:

Dodecyl methacrylate (k = 11) - 99.522,

2,2-Dimethoxy-2-phenylacetophenone - 0.41,

2,4-Ditretbutylorthoquinone - 0.068.

A matrix is prepared for cell transplantology, as in Example 1.

Characteristics are given in table 1.

Table 1 Qualitative assessment of the nature of the matrix-cell interaction Characteristics Example 1 Example 2 Example 3 Example 4 Example 5 Primary attachment in great numbers in great numbers in great numbers in small quantities in small quantities Location on polymer evenly evenly evenly evenly evenly Long-lasting Attachment there is there is there is no no The nature of growth, proliferation Intensive Intensive Intensive Slow Slow The number of connections between neurosphere-like
structures A lot of A lot of A lot of No No Monolayer formation To the 9th day To the 9th day To the 9th day Not visible Not visible

From table 1 it follows that in examples 1, 2, 3, in which the ingredients are taken in the amount corresponding to the claims, the matrix for cell transplantology has a high ability to attach planted cells, intensive growth and proliferation of cells, a large number of cell structures, the formation of dense monolayer cell network. Deviations from the method corresponding to the claims (examples 4, 5), lead to a matrix with low specified characteristics.

Recently, new methods based on the use of cell transplantology and tissue engineering to restore the lost structure or function of organs and tissues are increasingly being used. The implementation of the claimed matrix for cell transplantology is of great interest for medicine.

Claims (5)

1. A matrix for cell transplantology, characterized in that its main element is a flat plate made of a spatially cross-linked hydrophobic polymer containing hydrophilic groups and forming a layer of saturated hydrocarbons on the surface of the plate with a chain length of 8 to 18 carbon atoms oriented along normal to the surface of the plate. 2. The matrix for cell transplantology according to claim 1, in which a spatially cross-linked polymer is obtained by exposure to light with a wavelength of 320-380 nm photopolymerizable composition containing the ingredients:
Oligourethane methacrylate of the following structure:

where n is of the order of 35,
One of the methacrylic series monomers with a chain length of 8 to 18 carbon atoms of the following structure:
CH ₃ - (CH ₂ ) _k —O — C (O) —C (CH ₃ ) = CH ₂ , where k is from 7 to 17,
2,2-Dimethoxy-2-phenylacetophenone of the following structure:

2,4-Ditretbutylorthoquinone of the following structure:

while the above components are taken in the following ratio, wt.%:
Oligourethane methacrylate - 15-60,
The monomer of the methacrylic series with a chain length of 8 to 18 carbon atoms is 39-84,
2,2-Dimethoxy-2-phenylacetophenone - 0.4-3.0,
2,4-Ditretbutylorthoquinone - 0.01-0.06. 3. The matrix for cell transplantology according to claim 1, in which a spatially cross-linked polymer is formed on substrates with a hydrophobic surface. 4. The matrix for cell transplantology according to claim 1, in which the structure of the plate is perforated. 5. The matrix for cell transplantology according to claim 1 in the form of a volumetric element consisting of a multilayer complex of perforated plates.

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