Method for cell-free organic tissue preparation for living cell reimplantation and cell-free organic tissue produced by such method

FIELD: medicine.

SUBSTANCE: group of inventions refers to medicine, and can be used for production of transplantable tissues. A method for cell-free organic tissue preparation for living cell reimplantation involves cell-free tissue preparation on a substantially flat surface and creation of a number of holes on the surface, spaced along the whole surface mentioned above and arranged so that they penetrate deep down at least a portion of thickness of said tissue with the holes designed for keeping the reimplanted living cells. Said number of the holes is created by means of one or more metal needles connected to a power supply which on each needle point induces current flow of such intensity and of such waveform to provide adequate energy for destruction of molecular couplings comprising organic tissue near to the needle point mentioned above. Each hole is formed by current flow and is great enough that the needle point mentioned above could penetrate into a space formed by opening of the molecular couplings. The group of inventions also refers to cell-free organic tissue for living cell reimplantation produced by said method.

EFFECT: group of inventions enables reducing time of producing transplantation tissue and provides living cell penetration along the whole thickness of cell-free tissue.

24 cl, 4 dwg

 

The invention relates to a method for preparing acellular organic tissue for re living cells or "revival".

In medicine, specifically in the field of surgery, it is well known that the importance of tissue intended for transplantation in a living organism, to meet the growing need for replacement of parts of bodies in General.

Create biological substitutes that are prepared in the laboratory, and then are transplanted animals-recipients or people-recipients, is a medical technology known as "tissue engineering".

By known techniques of tissue for transplantation is prepared in the laboratory by implantation of cells into the matrix consisting of inorganic supportive environment, usually called "us" (cell frame or substrate).

The us is the material that is used to compensate for the loss of material of the body being treated, promotes spatial cell cultivation until then, until you form new tissue.

The us-material, of course, must then be subjected to decomposition to its complete disappearance and be replaced by synthesized cloth, aided cells implanted in the above-mentioned us-material.

Grafts mo is but to get this way or artificial, or us with natural materials (i.e. from the "donor"), which can be obtained from a person or animal, for example with the use of the esophageal wall.

To use natural us-material obtained from a donor for transplantation into another human body, the fabric should be primarily treated to eliminate all cells existing between the connective tissue fibers, and then to re human cells belonging to the intended recipient transplant ("owner"), in order to avoid rejection.

The technology used to create the us-material, i.e. acellular matrix, starting from tissue obtained from a donor, is well known and therefore not described in detail here briefly, they involve the immersion of tissue that must be processed in a liquid medium containing the enzyme substances able to "digest" and destroy the living cells contained in the tissue without damaging the connective fibers of the fabric.

After creating the matrix of the cell-free tissue, ready to receive cells from the recipient, the said fabric, or the us-the material is prepared in the so-called "Petri dish" (or similar container), which is a dish commonly used in laboratories for biological studies, at the bottom is toroi rests fabric for animation.

The revival of the tissue occurs through the implantation of stem cells from the future recipient and supply them with food through the culture medium, which nourishes the cells, keeping them alive and giving them the opportunity to multiply and disseminate.

Essentially, stem cells, initially placed on the upper surface of the fabric, pass through natural interstitium in the tissue of the us material - the interstitium, which was previously occupied by the donor cells.

After a specified period of time under controlled temperature and in the presence of nutrients that are contained in the culture medium, the cells repositioned in the interstitium of tissues, which thus becomes ready for transplantation into the body of the recipient.

It should be noted that cells that are typically used to revitalize the us-material, are stem cells, which then become differentiated (or already have those) and get specific function of the organ that is transplanted tissue, held a revival.

The success or failure of the transplant tissue treated in this way depends on the capillary diffusion cells through the matrix of the fabric. If this diffusion occurs with difficulty, or is carried out on the surface, but not deep, the recovery of tissue on the I transplantirovali becomes insufficient and begins the process of ossification, that leads to loss of the graft.

From the above it follows that to achieve success it is essential, if not necessary, to ensure the recovery of each tissue depth, particularly across its thickness.

Currently, even if the procedures for preparation and recovery are carried out on a sufficiently long period of time, it is impossible to provide results that are sufficiently reliable to ensure the success of the transplant.

This is due to lack of "penetration" of living cells, reimplanting in the us-material.

In practice, this drawback severely limits the possibilities for preparation of tissue suitable for transplantation because of thicker fabric does not fully pass recovery after transplantation because infiltration is carried out at an insufficient depth.

It follows that the currently used technology is applicable only for tissue transplantation is very limited thickness, for example, not more than about 0.1 mm

In U.S. patent 5112354 disclosed the preparation of bone allograft, which is first removed all soft tissue, and then the surface is structured to create a group of holes made with the possibility of a demineralization of the bone and increase the surface is La interactions with further input mesenchymal cells. Holes produced by laser or mechanical drilling.

The present invention is to develop a method for the preparation of cell-free tissue, allowing to overcome the above disadvantages.

More specifically, the present invention is to develop a method for the preparation of cell-free tissue, so that when the revival mentioned tissue stem cells mentioned cells easier penetrated in all possible space network of connective tissue fibers and colonized it to essentially restore the state of the tissue, characteristic of her before she became lifeless.

Another object of the invention is to achieve a significant and substantial reduction in processing time required for the cell-free recovery of the us material, after been added to living cells in order to prepare the tissue for transplantation.

These problems are solved using the method of preparation of the acellular organic tissue for recovery through re living cells, according to which:

- cook mentioned acellular tissue on the essentially flat surface;

- create a lot of holes on the surface of said fabric, distributed across said surface and arranged so that they pass the deep, at least a portion of the thickness of said fabric, these openings are provided for the contents of the said living cells after their re,

And in which according to the invention mentioned many holes create by means of one or more metal needles connected with a power source, which is at the tip of each needle causes the current passing such force and with such a waveform, to provide sufficient energy for breaking bonds between molecules constituting the organic tissue near the tip of the said needle, each hole is formed by the passage of current, and is large enough so that the tip mentioned needle could enter the space formed by the opening mentioned molecular bonds.

Preferably in the aforementioned one or more metal needles serves the electrical energy of alternating current, essentially sinusoidal with a frequency of about 4 MHz.

Preferably, the electric power supplied to the aforementioned one or more needles, contains harmonics, at least up to the third order.

Preferably the electrical voltage applied to the said one or more needles is about 200-230 C.

Preferably the current is passed through each needle is about 2-2,5 is A.

Preferably the depth of the mentioned holes corresponds to the full thickness of the aforementioned organic tissue.

Preferably the length of the above-mentioned holes greater than the thickness mentioned acellular organic tissue.

Preferably mentioned are many of the holes feature in a direction essentially perpendicular to the surface of said tissue or inclined to the surface of the aforementioned organic tissue.

Preferably one or more holes on the surface of acellular organic tissue is suitable for: content cells, designed to revitalize mentioned fabrics, is carried out using the device, which contains:

mentioned one or more metallic needles that are installed in the holder;

the source of electrical energy representing the said power supply and coupled with said one or more needles, suitable for applying current to the tip of each needle, the force and the wave form of which is such that it provides sufficient energy to result in breaking the bonds between molecules in the organic tissue in contact with the tip mentioned needle.

Preferably the said device contains many needles installed in the holder, capable of forming a matrix of rows of needles, essentially parallel to each other, being the m mentioned needle essentially equidistantly each other.

Preferably the diameter of the mentioned at least one needle is at least 50-55 microns.

Preferably the diameter of the mentioned at least one needle is greater than the maximum cell size for animation.

Preferably the said device comprises means for moving the aforementioned at least one needle along the three axes of a Cartesian coordinate system, i.e. along the vertical or inclined axis and along the Cartesian axes parallel to the surface of the organic tissue.

Preferably the tool movement creates movement along two Cartesian axes, which are located essentially parallele surface mentioned organic tissue.

These objectives are also addressed through the creation of acellular organic tissue, animated by re living cells, which is obtained using the described method.

Further characteristics and details of the invention will be illustrated in more detail in the description of the preferred variant of the invention, which is represented here as a non-limiting example, as well as devices that can be used in this way.

The invention is described below using the accompanying drawings, in which:

Figure 1 - diagram of the device in cross-section, containing the holder with a matrix of needles, resting on a layer of fabric, made the Noi for recovery;

Figure 2 is a view of one of the needles in the holder;

Figure 3 is a layout view of a matrix of needles;

4 is a diagram of the device for moving the needle holder.

According to the invention the pre-treated acellular organic tissue, the so-called us-the material is located on the bottom of Petri dishes (or similar container) so that it lies in expanded form on a flat surface.

Many of needles, such as needle marked position 1 and shown in figure 2, are arranged in a matrix, for example, forming a square, indicated generally by the position 3 in figure 3, so as to ensure the orderly arrangement of the needles, which are preferably separated by the same distance from each other, i.e. equidistantly relative to each other.

The head 11 of each needle 1 has an electric connection, for example, by a conductive metal plate 2 fixed to the holder 20 mentioned matrix needles. Mentioned plate 2 is connected with the electric wire 21, which, in turn, served the output voltage of the generator 4.

The mentioned generator 4 is the generator voltage, preferably generating a voltage 200-230 V, but with a frequency of 4 MHz, obtained with the use of electronic circuits, which are well known, so for the sake of brevity are not described here.

Sinusoidal voltage change at the output 1 of the generator 4 is preferably a distorted wave, and therefore, has harmonics at least first, second and third order.

The power generator 4 is regulated so that at the end of each electrode 1, it was possible to get power from 2 to 2.5 mA.

When the tip of each needle is resting on the surface 51 of the organic fabric 5, the contact between the tip of each needle and organic fabric gives you the ability to pass current of about 2-2,5 mA, as mentioned earlier. Mentioned current transfers energy to the surrounding molecules, which corresponds (as shown experimentally) that leads to the so-called phenomenon of "molecular resonance". This energy is sufficient only for breaking bonds between molecules that are affected by the passage of current, while in the surrounding area it causes no damage, tears, necrosis, decrease or increase thickness, changes in the content of the liquid phase, coagulation or other degradation of the fabric.

Essentially, such a gap formed in the molecular relations, is equivalent to the formation of tiny holes, which almost has the same diameter as the diameter of each needle 1, i.e. at least 50-55 microns.

Of course, can be used with needles of different diameter, larger or smaller, provided that the user does not forget about the fact that the diameter of the at least one needle exceeds the maximum size used CL the weave.

Further, the holder 20 of the matrix 3 of the needle is pushed in the direction indicated by the needle, and moving slowly enough so that as it moves the needle forward tip of the needle finds the hole that is already generated by the current flow and subsequent destruction of molecular bonds.

It is easy to see that, thus, no rupture of the connective tissue, and, therefore, creates a thin hole corresponding to the diameter of the insertion needle.

As previously explained, it is especially important and useful, because cells reimplantation on the fabric, can penetrate through the tissue and implanted on the walls of the holes, where they can multiply very quickly the recovery of organic tissue throughout the thickness.

As shown in figure 1, the needle 1 penetrate into the surface 51 us-material 5 preferably, but not necessarily inclined to increase the length of the holes, and consequently, to obtain the maximum effect of the permeability of the us-material.

Experiments have shown that for reviving a more efficient angle of 60 relative to the vertical, since the length of the resulting hole is greater than the thickness of the us-material.

Laboratory experiments showed that the effective matrix size 3 needles, containing needle 1 is about 1 cm2while the above matrix contains PR is approximately 200 needles; in this case, the current provided by the generator 4, does not exceed 500 mA.

Of course, the procedure of perforation should be repeated over the entire surface 51 us material to obtain a uniform distribution of holes through the thickness and across the useful surface of the tissue for transplantation.

For this purpose, the invention uses a device for creating a hole, which is preferably provided with a means 30 for moving the holder 20 along three axes of a Cartesian coordinate system, i.e. along the vertical or inclined to the Z axis and along the Cartesian axes X and Y parallel to the plane surface 51 and is schematically shown in Figure 4.

When in a given part of the us-material 5 holes are made, the holder 20 can be moved and the procedure can be repeated in an orderly manner to cover the entire surface 51.

It is clear that if the holder 20 carrying a matrix of 3 needles, connected to the programmable means 30 moves, for example using stepper motors controlled by an electronic control unit, the procedure can be applied automatically, consistently and with maximum precision.

After creating a series of holes in a cell-free tissue 5, as explained above, it is obvious that the above cell-free cloth can be placed in a Petri dish or similar container, where ZAT is m can be added to living cells, which usually represent the stem cells from the recipient, which is the transplant.

With proper nutrition using a nutrient culture medium mentioned stem cells can quickly and easily take all the holes made by the needles 1, thereby ensuring the full and effective recovery of the entire tissue intended for transplantation.

It is obvious that the method according to the invention solves all the tasks of the invention, since it results in a perfect and efficient recovery and prevents any risk of adverse outcome of transplant.

In addition, the recovery process is much faster than when using known technology, and with very positive results.

Where technical features mentioned in any of the claims are accompanied by reference characters, these reference symbols are included with the sole aim to make the claims more clear, and, accordingly, such reference characters do not impose any restrictions on the interpretation of each element identified as an example of such reference characters.

1. Method of preparation of acellular organic tissue for re living cells, according to which:
- goto the Yat mentioned acellular tissue, essentially flat surface;
- create a lot of holes on the surface of said fabric, distributed across said surface and arranged so that they pass into, at least a portion of the thickness of said fabric, these openings are provided for the contents of the said living cells after their reimplantations, characterized in that the said set of apertures create by means of one or more metal needles connected with a power source, which is at the tip of each needle causes the current passing such force and with such a waveform, to provide sufficient energy for breaking bonds between molecules constituting the organic tissue near the tip mentioned needles, each hole is formed by the passage of current, and is large enough so that the tip mentioned needle could enter the space formed by the opening mentioned molecular bonds.

2. The method according to claim 1, characterized in that on said one or more metal needles serves the electrical energy of alternating current, essentially sinusoidal with a frequency of about 4 MHz.

3. The method according to claim 2, characterized in that the electric power supplied to the aforementioned one or more needles, contains harmonics, at least until the third poradek is.

4. The method according to claim 3, characterized in that the voltage applied to the aforementioned one or more needles is about 200-230 C.

5. The method according to claim 2, characterized in that the voltage applied to the aforementioned one or more needles is about 200-230 C.

6. The method according to claim 2, characterized in that the current flowing through each needle is about 2-2,5 mA.

7. The method according to any one of claims 1 to 6, characterized in that the depth of the mentioned holes corresponds to the full thickness of the aforementioned organic tissue.

8. The method according to any one of claims 1 to 6, characterized in that the length of the above-mentioned holes greater than the thickness mentioned acellular organic tissue.

9. The method according to any one of claims 1 to 6, characterized in that the said set of apertures is placed in a direction essentially perpendicular to the surface of said fabric.

10. The method according to claim 8, characterized in that the said set of apertures have inclined to the surface of the aforementioned organic tissue.

11. The method according to any one of claims 1 to 6, characterized in that one or more holes on the surface of acellular organic tissue, suitable for the content of the cells after their reimplantations, performed with the use of the device, which contains:
mentioned one or more metallic needle (1), set the ies in the holder (20);
the source of electrical energy (4), which referred to the power source and connected with the said one or more needles, suitable for applying current to the tip of each needle, the force and the wave form of which is such that it provides sufficient energy to result in breaking the bonds between molecules in the organic tissue in contact with the tip mentioned needle.

12. The method according to claim 11, characterized in that the said power source consists of a generator, essentially sinusoidal voltage with a frequency of about 4 MHz.

13. The method according to claim 11, characterized in that the said device contains many needles installed in the holder (20), capable of forming the matrix (3) of the rows of needles, essentially parallel to each other, and the said needle, essentially, equidistantly each other.

14. The method according to claim 11, characterized in that the diameter of the mentioned at least one needle is at least 50-55 microns.

15. The method according to claim 11, characterized in that the diameter of the mentioned at least one needle is greater than the maximum size of the cells to re.

16. The method according to item 13, characterized in that the said needles mounted essentially perpendicular relative to said holder (20).

17. The method according to item 13, characterized in that the holder (20) in Berlin is located at an angle to the surface profile of organic tissue.

18. The method according to claim 11, characterized in that the said device comprises means (30) for moving the aforementioned at least one needle along the three axes of a Cartesian coordinate system, i.e. along the vertical or inclined axis (Z) and along the Cartesian axes (X, Y)parallel to the surface of the organic tissue.

19. The method according to p, characterized in that the said means of displacement creates moving along two Cartesian axes, which are located essentially parallel to the surface of the mentioned organic tissue.

20. The method according to claim 11, characterized in that the depth of the holes corresponds to the full thickness of the organic tissue.

21. The method according to claim 20, characterized in that the said set of apertures passes in a direction essentially perpendicular to the surface of the fabric.

22. The method according to claim 11, characterized in that the length of the holes is greater than the thickness of acellular organic tissue.

23. The method according to item 22, characterized in that the said set of apertures passes at an angle to the surface of the organic tissue.

24. Acellular organic tissue, prepared for re living cells, characterized in that it is obtained by the method according to any of the preceding paragraphs.



 

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1 ex

FIELD: medicine.

SUBSTANCE: bone of natural origin is cleaned, sawed up to 0.2-2.0 cm thick plates, washed with heated to 65°C 0.1 M pH 5.8-6.0 phosphate buffer, digested in 0.1-0.4% activated papain solution at 65°C during 24 hours, then washed in five volumes of water at 40-80°C, treated with 0.4 N alkali at room temperature during 10-24 hours, rinsed in running water, degreased in ethanol/chloroform mixtures in ratio 1:2 firstly, and 2:1 secondly, decalcified in 0.4-1 N hydrochloric acid, treated with 1.5-3% hydrogen peroxide during 4 hours, washed with purified water, then with ethanol, dried at room temperature, packed up and sterilised. Material for osteoplasty and tissue engineering represents compound, in which native collagen matrix space structure and natural bone mineral component are preserved, containing 25% collagen and 75% mineral matter. According to dry material analysis it includes less than 1% non-collagen proteins.

EFFECT: method improvement.

3 cl, 5 ex

FIELD: medicine.

SUBSTANCE: method involves chemical stabilisation of biotissue with 0.625 % aqueous solution of glutaraldehyde, pH 7.4, followed with preparation with a surface-active substance and quadruple change of a working solution. Immediately ahead of implantation, bioprostheses are thoroughly washed with sterile physiologic saline sixfold changed, at 500 ml of the solution for 100 g of biotissue. Then it is processed with 0.05-0.5% aqueous solution of chitosan N-sulphosuccinate of molecular weight 50-150 kDa in intensive stirring during 0.5-2 h with pH within 5 to 8, and to temperature 222C. Further, it is fixed in sterile absolute ethanol and put in sterile physiologic saline, and stored at temperature 6-8C before implantation.

EFFECT: improved durability of bioprostheses.

5 ex, 4 tbl

FIELD: medicine.

SUBSTANCE: there is offered method for chemical treatment of xenopericardium that involves chemical stabilisation of xenopericardium with 0.625% glutardialdehyde and following processing with 1% sodium dodecyl sulphate; chemically stabilised xenopericardium is additionally processed with 0.050.25% aqueous solution of chitosan or metal-containing chitosan with deacetylation degree 5098% and molecular weight 4140103 at pH 35; upon termination of processing, xenopericardium is fixed in 70% aqueous solution of ethanol; then modified xenopericardium is kept in 0.100.50% aqueous solution of chitosan N-sulphosuccinate with molecular weight 10166103 or chitosan 3,6-O-disulphate with molecular weight 7180103 at pH 48 during 2060 mines at temperature 2030C with following fixation in absolute ethanol.

EFFECT: improved durability and biocompatibility of bioprostheses.

3 ex, 7 tbl, 3 dwg

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