WO2003080141A1

WO2003080141A1 - Biomatrix and method for producing the same

Info

Publication number: WO2003080141A1
Application number: PCT/DE2003/000904
Authority: WO
Inventors: Thomas Graeve
Original assignee: Ars Arthro Ag
Priority date: 2002-03-21
Filing date: 2003-03-19
Publication date: 2003-10-02
Also published as: DE10391674D2; EP1485139A1; AU2003223880A1; JP2005520638A

Abstract

The invention relates to a biomatrix and to a method for producing the same. The aim of the invention is to provide a prosthesis with a satisfactory biological and mechanical action, for example for diseased cartilage. To achieve this, the biomatrix is compressed or compacted. The invention also relates to a corresponding method.

Description

Biomatrix and process for its production

The invention relates to a biomatrix and a method for its production.

In medical technology, especially in surgery, the task is regularly to remove defective components on the human or animal body and to replace them with artificially produced ones. Medical technology is the subject of intensive research. It has been shown that many special solutions are required for the various different fields of activity.

For example, in the field of repairing joint diseases, DE 100 26 789 AI describes a cartilage replacement that allows good regeneration of a treated cartilage defect. The application mentioned makes use of a 3D biomatrix that can be adapted in shape and size to the defect to be replaced ,

The state of the art also includes many other suggestions for implants in the area described. The materials used here are either three-dimensional, but porous, for example in the case of a configuration as a sponge or fleece; or they are solid, but have no three-dimensional structure, for example in the case of an embodiment as a film. However, special requirements must be placed on implants that are to replace parts of the mechanical body structure. On the one hand, a defect to be repaired generally has at least a certain three-dimensionality, so that the implant should also be three-dimensional. On the other hand, due to its density or, in particular, its pressure stability, the implant must be able to take into account the mechanical loads when used in the body structure. In particular, the pressure stability of the previously known materials is not sufficient for use, for example, in the cartilage area.

The inventor has also set himself the task of developing a Biomatrix so that the Biomatrix as an implant can also cope with the high loads that occur in mechanical matters. In addition, the inventor has set himself the task of developing a method with which such a biomatrix can be produced.

The task is solved to a surprisingly good degree by a biomatrix in which the matrix is compressed or solidified.

A collagen biomatrix as described in DE 100 26 789 AI can be three-dimensional per se. By compressing or solidifying the collagen biomatrix, the biological material becomes so strong that the dynamometric requirements in the mechanical body structure can be met. The compressive strength of the compressed or solidified material in particular can meet very high demands. But tensile strength also increases compared to known biomatrices. ge with an increasing resistance to transverse contraction associated with the higher compressive strength.

The invention thus opens up far-reaching uses for implants, for example as cartilage replacement with high compressive strength, ligament replacement, selmen replacement, meniscus replacement, ligament disc replacement, nucleus replacement and / or annulus replacement. Such an implant can even be used as a bone replacement or as a replacement for bone-cartilage constructs.

The biomatrix compressed or solidified according to the invention can advantageously be characterized in that it has a density gradient. The biomatrix can be homogeneously compressed, but the collagen structure can also be produced in gradient form. As a result, the resulting implant can be adapted in a special way to the defect on the body trunk in accordance with the dynamometric requirements. It is also possible to economically manufacture implants with reinforced areas for special requirements.

In a preferred variant, the density gradient is stepped. A biomatrix according to the invention is generally relatively easy to produce with a step-shaped density gradient.

Alternatively, the density gradient can also have a continuous transition. A biomatrix designed in this way is relatively complex to manufacture, but can be adapted to the mechanical requirements at a defective location to an outstanding degree. By a density gradient with a continuous transition, an implant can fit particularly homogeneously into the surrounding body structure, without - as with the discontinuous density gradient of the implant and continuously changing properties of the neighboring structures - no harmful voltage peaks.

The biomatrix according to the invention can particularly preferably be distinguished in that the density gradient forms a body with a firmer outer shell and a softer core. A softer core is also understood to mean a much softer core, including a core without its own hardness. For example, the core can be filled with a fluid or in particular a gas, so that a mechanical effect similar to that of an air cushion is achieved. As a result, the properties of the implant are particularly well matched to the neighboring structures. The soft core ensures sufficient flexibility of the Biomatrix when it is in use, while the solid outer shell protects particularly well against mechanical damage to the Biomatrix. In this way, the Biomatrix can still be sufficiently elastic with a long service life and great robustness.

It goes without saying that a biomatrix with a density gradient is also advantageous and inventive, regardless of an explicit compression or consolidation of the biomatrix.

Regardless of this, it is advantageous if the matrix according to the invention is combined with inert materials. The inert materials can be of both biological and non-biological origin. Collagen fleeces are mentioned here as examples.

In a preferred embodiment, the biomatrix according to the invention has aligned fibers, in particular collagen fibers. The combination of compression and simultaneous application of changing pressure and tensile loads enables the collagen fibers to be aligned in the matrix. Alternatively and cumulatively, this is also possible by applying physical AC voltages or by other physical methods. Aligned fibers have a particularly advantageous effect on the physical properties of the biomatrix. If collagen fibers are used for this, both the physical and the biochemical properties experience a significant improvement.

It is understood that the presence of aligned fibers, in particular collagen fibers, in the biomatrix can also be achieved without explicit compaction or consolidation and is also advantageous and inventive independently of this.

Finally, an embodiment variant of the biomatrix according to the invention is particularly preferred in which the starting solution has 1 to 10 mg protein per ml gel solution and this starting solution is compressed to more than 50 times the original protein content, preferably more than 50 times. The protein content can even up to Hundred times the original protein content. This creates a very pressure-stable matrix with high strength.

The task also solves a method for producing a biomatrix, in which the matrix is compressed or solidified. By compressing or solidifying the Biomatrix achieves the advantageous properties mentioned above,

The method according to the invention is preferably characterized in that the matrix is produced using a method for redifferentiating and / or multiplying dedifferentiated cartilage cells, in which undifferentiated cartilage cells are embedded in a three-dimensional, gel-like biomatrix containing at least 1.5 mg / l collagen in buffered serum-containing cell culture medium, to be cultured. The main problem of degenerative or traumatic joint diseases is that the damaged articular cartilage shows only a slight ability to regenerate. A major task therefore consists in permanently inserting cartilage cells with the implant at the site of the defect enable the cartilage to regenerate. If the matrix is produced using the described method, the dedifferentiated cartilage cells in the biomatrix can redifferentiate and resume their cell-typical metabolic performance. For this purpose, the biomatrix should contain a collagen structure newly composed of a preferably fresh collagen solution with a concentration of at least 1.5 mg collagen per ml biomatrix. In connection with the present invention, the term “culturing cells” is understood to mean maintaining the vital functions of cells in a suitable environment, preferably taking place in vitro, for example by supplying and removing metabolic educts and products, in particular also an increase of cells, Cartilage cells are understood to mean naturally occurring or genetically modified cartilage cells or their precursors, which can be of animal or human origin.

In a particularly preferred variant, the process according to the invention is characterized in that the matrix is produced by a process comprising the isolation of collagen-containing tissue

Transferring the collagen-containing tissue into acidic solution, incubating the collagen tissue transferred into the acidic solution at 2 to 10 ° C., in particular 4 ° C., centrifuging undissolved collagen portions, mixing the resulting collagen solution at 2 to 10 ° C., preferably 4 ° C, with a solution containing cell culture medium, serum and buffer, and gelling the mixed solution by increasing the temperature. This method is very effective and can provide enough cell material for the production of cartilage grafts even with a small amount of cartilage tissue. In addition, a homogeneous distribution of the cells in the biomatrix is achieved in a simple and reliable manner. The strength of the resulting material can be adjusted by the initial volume of the collagen solution. Depending on the application, it can be advantageous if the matrix is produced with cells in the method according to the invention. The compressed or solidified material can be produced with or without cells. The production with cells supports the biocompatibility of the resulting implant in the manner described.

In an alternative to this, the matrix according to the invention is produced without cells. This is particularly useful when there are no suitable cells available to produce the matrix. In addition, the resulting implant is simpler and cheaper to manufacture, but also meets the requirements for mechanical strength.

It is advantageous that, in the method according to the invention, the compression is effected by withdrawing liquid. If the matrix is one

If there is an excess of liquid, then the removal of this excess liquid is the easiest way to compress or solidify the matrix. There are various possibilities for practical implementation here. The matrix can be pressurized physically, for example, or a negative pressure can be applied. Liquid can also be reliably extracted from the matrix using capillary forces or a combination of the described possibilities. In most cases, the easiest way to do this is to remove water. Such liquid withdrawal can be achieved particularly effectively by pressurizing or applying a negative pressure. Alternatively and cumulatively, it is advantageous if the compaction is brought about by chemical bonding. Chemical bonding represents a further possibility of removing water and / or other liquid from the biomatrix and thereby compressing or solidifying the biomatrix in order to achieve the desired properties.

In addition, the method according to the invention can also be used to produce a collagen thread with a diameter between 0.005 and 4 mm, preferably between 0.01 and 2 mm. Such a collagen thread opens up many new possibilities for insertion into the human or animal body. In particular, it can even have a connection functionality.

To produce such a thread, it is proposed that the collagen solution pass through a nozzle. Along with the narrowing of the cross-section in the nozzle, the biomatrix is also compulsorily compressed. For this purpose, the liquid collagen can be pressed through the nozzle with or without a buffer solution, which withdraws liquid from the collagen. This can be done by applying positive or negative pressure. After compaction, the collagen thread can be dried, whereby a proportion of approx. 10% residual moisture is readily permitted.

Methods according to the invention are explained below using two examples. However, it should be emphasized here that the present patent application not only relates to cartilage cells, as in the example, but also to bone cells, stem cells, fibroblasts, endothelial cells, muscle cells, epithelial cells, glandular cells, sensory cells and combinations the cells mentioned. The invention can also easily be applied to these cell types,

example 1

A collagen solution, as described in DE 100 26 789 AI, with a protein concentration of 6 mg / ml gel solution serves as the starting material. At the same time, a buffer solution is used to buffer the acidic collagen solution,

The two liquids are combined in equal parts at 4 ° C and mixed together. The mixed solution therefore has a collagen concentration of approximately 3 mg / ml. This solution is now filled into a glass cylinder with a diameter of approx. 2 cm. In the present case, approx. 35 ml of collagen buffer solution mixture are filled into the cylinder, resulting in a filling height of approx. 10 cm.

The lower part of the glass cylinder is in a glass cover, which ensures a reliable seal. In addition, the lower end of the glass cylinder is closed with a porous, liquid-permeable membrane.

The collagen buffer solution mixture is incubated in the cylinder for 20 minutes at 37 ° C., during which it gels completely. A pressure stamp is then applied to the gelled matrix, which is inserted through the upper, open end of the glass cylinder and the matrix pressurized along the cylinder axis. The pressure here is approximately 0.2 Pa and is maintained for 20 minutes.

The pressure compresses the matrix and squeezes liquid out of it. This happens first at the upper end of the matrix, to which the Daick is originally applied; a short time later there is also a liquid outlet at the lower opening which is closed by the liquid-permeable membrane. The pressure stamp is carried along with the compressed matrix in order to maintain the applied pressure as unchanged as possible,

As the matrix becomes denser, that is to say as the height of the matrix column decreases, the protein content of collagen increases and the biomatrix solidifies according to the invention. After the pressure has been applied, the pressure stamp is removed and the glass cylinder is removed from the base. The leaked liquid simply flows away, the compressed matrix remains. In the present experiment, the matrix still has a height of approx. 2 cm after compaction, the protein content has quintupled compared to that in the starting mixture and is approx. 15 mg / ml. With further compression, protein contents of collagen of, for example, 30 mg / ml gel and more can also be easily achieved,

The compressed matrix is then mechanically cleaned and opened, for example cut, according to the later application. The graft is then ready for cultivation. The wall of the standing cylinder can consist of non-porous material, for example glass, or of porous material, for example an ultrafiltration membrane. The shape of a cylinder is not binding here, the compression or consolidation of the biomatrix can also take place in any other shape, for example also in the shape of a sphere or in the form of a strand. The compressed matrix is shaped in accordance with the later application.

However, the compressed matrix can also be adapted to the later shape by mechanical methods such as cutting or punching and by physical methods, for example lasers. The diameter and the thickness of the compressed matrix are variable. The diameter can range from 1 mm to 200 mm, the thickness from 1 mm to 50 mm.

Example 2

Reference is made to the drawing in order to clarify this example of a process according to the invention. Here shows

the only figure schematically shows an experimental setup for the production of collagen fibers.

A collagen solution 1 as described above is again used as the starting material. This is pressed by a pump 2 via a line 3 a, 3 b through a nozzle 4 with a cylindrical section 5 and a conical section 6. Optionally, an auxiliary pump 7 can be used to Council container 8 and a buffer solution 9 are introduced into the line 3b and are passed through the nozzle 4 together with the collagen solution 1.

As a result of the narrowing of the cross-section in the conical section 6 of the nozzle 4, the collagen solution undergoes a strong compression, in which the liquid is extracted from it (in the figure illustrated by the arrows numbered 10 as an example). At an outlet opening 11 of the nozzle 4, a collagen thread 12 emerges, which essentially has the average of the outlet opening 11. The collagen thread 12 can then be dried

Claims

claims:

1. Biomatrix, characterized in that it is compacted or solidified.

2. Biomatrix according to claim 1, characterized in that it has a density gradient

3. Biomatrix according to claim 2, characterized in that the density gradient is step-shaped.

4. Biomatrix according to claim 2, characterized in that the density gradient has a continuous transition,

5, biomatrix according to one of claims 2 to 4, characterized in that the density gradient forms a body with a firmer outer shell and a softer core.

6. Biomatrix according to one of the preceding claims, characterized in that the matrix is combined with inert materials.

7. Biomatrix according to one of the preceding claims, characterized in that it has aligned fibers, in particular collagen fibers.

8, biomatrix according to one of the preceding claims, characterized in that the starting solution has 1 to 10 mg protein per ml gel solution and this starting solution on the Zelnfa before, preferably more than fifty times, the original protein content is compressed.

9. Process for producing a biomatrix, characterized in that the matrix is compressed or solidified.

10. Verfaliren according to claim 9, characterized in that the matrix is produced with a method for redifferentiation and / or propagation of dedifferentiated cartilage cells, in which dedifferentiated cartilage cells embedded in a three-dimensional, gel-like biomatrix containing at least 1.5 mg per liter collagen in buffered cell culture medium containing serum, can be cultivated.

11. The method according to claim 9 or 10, characterized in that the matrix is produced according to a process comprising isolating collagen-containing tissue, converting the collagen-containing tissue into an acidic solution, and incubating the overfilled into the acidic solution Collagen tissue at 2 to 10 ° C, especially 4 ° C, centrifuging undissolved collagen portions, mixing the resulting collagen solution at 2 to 10 ° C, preferably 4 ° C, with a solution containing cell culture medium, serum and buffer, and gelling the mixed solution by increasing the temperature.

12. Verfaliren according to any one of claims 9 to 11, characterized in that the matrix is made with cells.

13. Verfaliren according to any one of claims 9 to 11, characterized in that the matrix is made without cells.

14. The method according to any one of claims 9 to 13, characterized in that the compression is effected by withdrawing liquid.

15. Verfaliren according to claim 14, characterized in that the

Fluid withdrawal is initiated by pressure surges.

16. According to claim 14, characterized in that the

Fluid withdrawal is carried out by applying a vacuum.

17. Verfaliren according to any one of claims 9 to 16, characterized in that the compression is effected by chemical bonding.

18. The procedure according to one of claims 9 to 17, characterized in that a collagen thread with a diameter between

0.005 and 4 mm, preferably between 0.01 and 2 mm.

19. According to claim 18, characterized in that the collagen solution passes through a nozzle to form the collagen thread.

20. According to claim 18 or 19, characterized in that the collagen thread is dried after the compression