Methods, instruments and materials for transplanting cartilage tissue cells

FIELD: medicine.

SUBSTANCE: method involves producing and transplanting and implantable segment containing mature cartilage tissue cells fixed on absorbable supporting matrix for repairing animal cartilage. The implantable segment has absorbable elastic supporting matrix for culturing and fixing living cells thereon. Instrument for introducing the implantable segment, having mature cartilage tissue cells on supporting matrix, into defective animal cartilage area, has clamps and external tubular envelope. The envelope has an end holdable by user and an end for making introduction into defective cartilage area. Holder and telescopic member are available in the envelope end holdable by user. Injection canal is partially embedded into the holder and projects beyond the holdable envelope end towards the end for making introduction. The clamps are attached to the telescopic member. They are well adapted for catching and releasing the implantable segment when telescopically moving the holder in the envelope.

EFFECT: enhanced effectiveness in arranging and fixing implantable segment in the implantation place.

47 cl, 11 dwg

The present invention relates to the field of transplantation of Mature cartilage cells, transplantation and treatment of bone and cartilage, repair joints and prevention of arthritic pathologies. In particular, the present invention is directed to new methods and tools for the transplantation of Mature cells of the cartilage and restore cartilage.

In the United States annually, more than 500,000 operations arthroplasty and replacement joints of all kinds. Approximately the same number of operations performed in Europe, including approximately 90,000 replacement surgery of the knee and about 50,000 operations to eliminate defects in the knee. Approximately the same number of such operations performed in the U.S. (Praemer, A., Fumer, S., Rice D.P. Musculoskeletal conditions in the United States, American Academy of French Brasserie Surgeons, Park Ridge, III, 1992, 125).

Method of regenerative treatment of cartilage would be most applicable and could be conducted at an early stage of damage to the joints, thus reducing the number of patients needing surgery to replace joints. When such preventive treatments will diminish and the number of patients developing osteoarthritis.

The methods used to change the structure of the cartilage in the joints, mainly aimed at restoring cartilage using drilling and curettage t the Ani in the subchondral region, which removes the diseased cartilage and Podhradie bone, exposing the mesh, surrounded by blood vessels bone (Install, J. Clin. Orthop. 1974, 101, 61; Ficat R.P. et al., Clin. Orthop. 1979, 144, 74; Johnson L.L., in Operative Arthroscopy, McGinty J.B., Ed., Raven Press, New York, 1991, 341).

There is a method of culturing cells for synthesizing cartilage from embryological chicken (Coon and Cahn, Science, 1966, 153, 1116).

Later, Kahn and Laser (Cahn and Lasher PNAS USA 1967, 58, 1231) used the analysis system disorders of DNA synthesis that is required as a prerequisite for differentiation of cartilage. Mature cartilage cells corresponded as EFG and FGF growth (Gospodariwicz and Mesher, J. Cell discrimination) 1977, 93, 117), but completely lost the ability to differentiate (Benya et al., Cell 1978, 15, 1313).

The method of growing Mature cartilage cells have been described principally used with small improvements over Brittberg (Brittberg, M. et al. New Engl., J. Med. 1994, 331, 889). Grown in this way cells are used as autograft in the knee joints of patients.

In addition, Kolletas et al. (J. Cell Science 1995, 108, 1991) have studied the expression of specific molecules of substances cartilage, such as collagen and proteoglycans, during prolonged cultivation of the cells. It was found that, despite the morphological changes in single-layer cultures in the process of cultivation (Aulthouse, A. et al. In vitro Cell Dev. Biol., 1989, 25, 659, Archer, S. et al., J. Cell Sci. 1990, 97, 361; Hoenselann, H. et al., J. Cell Sci. 1994, 107, 17; Bonaventure, J et al., Exp. Cell Res. 1994, 212, 97), compared to suspension cultures grown in the gels agarose, drops of alginate or culture cocoon (preserving the morphology of round cells), proven by many scientists remained unchanged markers expression of Mature cells of the cartilage, such as collagen types II and IX, and the large aggregating proteoglycans, aggrecan, versican and binding protein (Kolettas, E., et al., J. Cell Science 1995, 108, 1991).

Know the use of gels of collagen type I in experiments with animals for repairing cartilage (Wakitani et al., Tissue Engineering, 4 (4), 429, 1989).

In all cases, the main problem was the lack of biomechanical properties required for functional tissue repair.

Mature cartilage cells are specialized cells derived mesenchyme, which are located exclusively in the cartilage. Cartilage is an avascular tissue, the physical properties which depend on intercellular substance, which produce Mature cells of cartilage. In the process endochondral of ossification of cartilage cells Mature, leading to cellular hypertrophy, characterized by the manifestation of the expression of collagen type X (Upholt, W.B. and Olsen, RR, in: Cartilage : Molecular Aspects (Hall, B. & Newman, S., Eds) CRC Boca Raton 1991, 43; Reichenberger, E. et al., Dev. Biol. 1991, 148, 562; Kirsch, I. et al., Differentiation, 1992, 52, 89; Stephens, M. et al., J. Cell Sc., 1993, 103, 1111).

In addition, a pronounced degradation of collagen type II in the outer layers of the cartilage surfaces of the joints caused by osteoarthritis. The network of collagen correspondingly thinner and after that develops fibrillazione, resulting in losses and the displacement of the matrix substances such as proteoglycans. This fibrillazione osteoarthritis cartilage can bring to obyzvestvleniya cartilage and subchondral bone (Kempson, G.E. et al., Biochem. Biophys. Acta 1976,428, 741; Roth, V., and Mow, V., J. Bone Joint Surgery, 1980, 62A, 1102; Woo, S.L-Y, et al., in Handbook of Bioengineering (R. Skalak and S. Chien, Eds), McGraw-Hill, New York, 1987, pp.4.1-4.44).

Describe the basic design, histology and microscopic anatomy of bone, cartilage and other connective tissue can be found, for example, the Wind Burkitt and Daniels "Functional histology" (Wheater, Burkitt and Daniels, Functional Histology. 2^ndEdition (Churchill Livingstone, London 1987, Chp.4). In addition, describe the basic histological anatomy of bone defects, cartilage and other connective tissue can be found, for example, the Wind, Stevens and Lowe General histopathology" (Wheater, Burkitt, Stevens and Lowe, Basic Histopathology. (Churchill Livingstone, London 1985, Chp.21).

Although the need for transplantation of cartilage cells has been described in at least the limits specified references, the question about the necessity of developing a satisfactory and efficient way Voss is the resolution of cartilage by transplantation or otherwise remains open.

The present invention is directed to the creation of the implantable segment, comprising a supporting matrix, which can support the growth and attachment of cells, and a method of implantation of such a segment to restore the cells in the site of implantation. In one embodiment of the present invention, a method for effective treatment of the surfaces of the articular cartilage surfaces in animals by transplantation of implantable segment, including Mature cells of the cartilage, held-absorbable support matrix. In one embodiment of the invention, the support matrix is made of collagen type I or type II, and Mature cartilage cells are autologous or homologous. Preferably implantable segment attached to the transplanted area with adhesive or mechanical fastening element.

In addition, the present invention is directed to an instrument for the introduction of the implantable segment, which is designed for placement and adjustment of the implantable segment at the site of implantation, as well as element for the fixation of the implantable segment of the graft site.

Moreover, the present invention is directed to the creation of the implantable segment to restore cartilage in animals, including Mature cells of cartilage tissue and, held absorbed in the support matrix, and the manufacturing method.

The present invention is illustrated in the following drawings.

On figa shows the normal articular end of the bone in the knee joint with the surface of the joint cartilage covering the articular end of the bone.

On FIGU shows the cartilage defect or injury of the cartilage covering the articular end of the bone.

Figure 2 shows one embodiment of an implantable segment according to the present invention.

Figure 3 shows the location of the arc implantable segment according to figure 2 in the tool for the introduction of the implantable segment prior to implantation, for example, as shown in figure 4.

Figure 4 shows a tool for the introduction of the implantable segment for implantation in a defective area of the cartilage tissue according to the present invention.

Figure 5 shows a diagram of the location of the implanted segment, according to figure 3, in place of the defect or injury in the cartilage surface with two input channels, in which you can enter a tool for the introduction of the implantable segment.

Figure 6 shows a schematic cross section of cartilage defect or an injury that does not extend to the subchondral layer, and implanting the segment, according to this invention, attached to the site of the defect or injury.

7 showing the cross section of a cartilage defect or injury, which does not extend to the subchondral layer, and implanting the segment according to this invention, attached to the site of defect or injury mechanical locking climera.

On Fig shows one embodiment of a mechanical fixing of climara used for fastening the implantable segment to the area of the defect or injury.

Figure 9 shows a cross-section of cartilage defect or injury, which is distributed in the subchondral layer, and implanting the segment according to the present invention attached to a defective or injured area.

Figure 10 shows a cross-section of cartilage defect or injury, which is distributed in the subchondral layer, and implanting the segment according to the present invention, attached to the site of defect or injury mechanical locking climera.

On figa shown black-and-white copy of a color micrograph of histological samples of solid support matrix at the beginning of growth on it and cartilage cells.

On figa shown color micrograph figa.

On FIGU shown black-and-white copy of a color micrograph depicting the support matrix on figa filled with cartilage cells after three weeks of cultivation on it.

On figw presents a color micrograph figv.

On IGS presents a picture of the reference matrix, formed by collagen with Mature cartilage cells grown on it and shows immunohistochemical staining.

On fig.11D presents a photograph of the reference matrix formed from collagen and having a Mature cartilage cells grown on it in the bioreactor system and identified by immunohistochemical staining.

The invention is as follows.

As stated above, one of the joints of the human body, which often happens trauma or defect of cartilage, is the knee joint. On figa shows a common connection end of the bone in the knee joint of the person. The knee joint 10 is formed by the connection of the femur 12 and balyshevatravel 14 bones, healthy cartilage 16 covers the articular end of the femur 12. On FIGU shown marked around the area of the defect or injury 18 (hereinafter defect 18) in the cartilage 16.

The present invention includes a regenerative cartilage implant, the method of implantation and implantation. The implant includes a support matrix and autologous and homologous cells held therein.

In General, the support matrix is a material that should support the growth of cartilage cells and which must eventually be absorbed into the body of a patient subjected to implantation. Transaction Tran the plantation can be performed by arthroscopic, minimally invasive or open surgical technique.

Furthermore, the method of the present invention involves the use of a suitable allogenic and xenogenic cartilage cells to repair cartilage defects.

Figure 2 shows the implant. In particular, implantable segment 20 includes a support matrix that hold it in cartilage cells 24. Suitable support matrix 22 may be solid or gel-like skeleton, characterized by the ability to maintain a stable form within the time period of growth in him cartilage cells before and after transplantation and to establish a system similar to the natural environment the location of the cartilage cells, to optimize the differentiation of their growth.

The support matrix 22 must be stable for a period of time sufficient for full recovery of the cartilage, then the matrix is absorbed into the body, for example, within two months, leaving virtually no trace and toxic degradation products. The term "absorbed" means the processes by which the reference matrix decays by natural biological processes, and then spread out the support matrix and the decay products are displayed, for example, through the lymphatic or blood vessels. In this regard, the support matrix 22 is preferably made of physiologists who Eski absorbed panthenol material in the form of a membrane. In addition, preferably the support matrix has the form of a sheet with one relatively smooth side 21 and the other relatively rough side 23. Rough side 23, for example, is fibrous and usually covers the defect 18 cartilage and promotes the ingrowth of cartilage cells inside, and smooth side 21 usually turns away from the defect 18 cartilage, making it difficult to ingrowth of tissue.

In one embodiment of the support matrix 22 is formed from polypeptides or proteins. Preferably the polypeptides and proteins obtained from natural sources, for example from mammals. However, artificial materials, physical and chemical properties similar to natural, also apply for the formation of the support matrix 22.

Preferably, the support matrix 22 was elastic, because it is regulated by the user to perform manipulation with implantable segment 20 and then returns to its original shape, according to one embodiment of the present invention, as described below.

The preferred material for the formation of the support matrix 22 is collagen, taken, for example, a horse, pig, cow, sheep and chicken. Suitable materials from which it is possible to form the supporting matrix, are Chondro-Cell®, (commercially available in the form of a strip matrix of collagen type I, Ed. Geistlich Soehne, Switzerland) and Chondro-Gide® (commercially available in the form of a strip matrix of collagen type I, Ed. Geistlich Soehne, Switzerland). The support matrix 22 formed from collagen type I, more rigid than the support matrix 22 formed from collagen type II, although the latter can also be used.

Implantable segment, as described above, can be produced, for example, cultivating Mature cells of the cartilage on the support matrix, as will be described below in more detail.

For apologizes implant remove sampling cartilage by arthroscopic technique first from the field, not carrying the weight of the load in the joint of the patient and transferred to the laboratory in a nutrient medium containing 20% fetal calf serum. Then sampling cartilage treated with enzyme, for example trypsin - ethylenediaminetetraacetic acid (edtc), a proteolytic enzyme and a binding agent for separation and extraction of cartilage cells. Then extracted the cartilage cells are cultivated in a nutrient medium from the number of initial cells of about 50,000 cells until the final number about 20 million Mature cells of the cartilage, and more.

Three days before re-implantation of culture medium change on the transporting medium containing 10% autologous serum (namely, serum extracted from the blood of the patient is, as described below). Then cultured Mature cells of cartilage tissue in the transplanted environment absorbed and penetrate into the support matrix 22 and continue to multiply, forming the implantable segment 20, which is then implanted at the site of the cartilage defect 18 in a patient.

Defect or injury 18 can be processed directly, or slightly extending scalera surgically prior to implant for placement of implantable segment 20.

The process of culturing cells and nutrient medium and environment for transplantation are described in detail by examples below, starting with a description of the laboratory procedures of processing the extracted cartilage biopsy and cultivation of Mature cells of cartilage tissue according to the present invention.

The nutrient medium used for the treatment of cartilage biopsy in the process of growing and maturing cartilage cells, is produced by mixing together 2.5 ml of gentamicin sulfate (concentration 70 µmol/l)4,0 ml amphotericin b (trademark Fungizone®, antifungal drug, supplied from Squibb) - concentration of 2.2 mmol/l; 15 ml of 1-ascorbic acid (300 µmol/l), 100 ml of fetal calf serum (final concentration 20%), the rest DMEM/F12; thus receive about 400 ml of culture medium.

The same environment is used to transfer chrysiogenetes from the clinic to the laboratory, in which Mature cells of the cartilage tissue is extracted and propagated.

Blood taken from the patient, centrifuged at a rate of about 3000 rpm to separate the serum from the other components. Separated serum preserved and used in a later stage of the cultivation process and the transfer process.

Cartilage sampling, previously taken from the patient for autologous transplantation, is transferred in the above-described nutrient medium to the laboratory for further cultivation. Wednesday cultivation was filtered to separate cartilage biopsy and then thrown away upon arrival at the laboratory. Then the cartilage sampling washed in pure DMEM/F12 at least three times to remove the film of fetal calf serum cartilage biopsy.

After this cartilage sampling washed with the composition comprising the above-described environment of cultivation, to which add 28 ml of trypsin EDTA (concentration 0,055). In this part of the cartilage sampling incubated for five to ten minutes at 37°C and 5% CO₂. After incubation of cartilage sampling washed twice or thrice in a nutrient medium for the full treatment of biopsy material from any of the enzyme trypsin. Then the cartilage is weighed. Typically, the minimum amount of cartilaginous tissue that is required for growing cartilage cells, approximately 80-100 is, Preferred is a slightly greater number, for example, 200-300 mg

After weighing the cartilage is placed in a mixture of 2 ml of collagenase (concentration of 5000 enzyme units; (a digestive enzyme) in approximately 50 ml of pure medium DMEM/F12 and crushed for partial digestion of the cartilage of the enzyme. After that crushed the cartilage is transferred through the funnel into the bottle, adding there are approximately 50 ml collagenase and pure mixture of DMEM/F12. Then the crushed cartilage incubated for 17-21 hours at 37°C and 5% CO₃.

In one embodiment, the incubated crushed cartilage strain through a mesh size of 40 μm, centrifuged (at 1054 rpm or 200 times gravity) for 10 minutes and washed twice in culture medium. Then the Mature cells of the cartilage is considered to determine their viability, and then incubated in nutrient medium at 37°C and 5% CO₂at least two weeks, during which time the culture medium is changed three or four times.

Three days prior to re-implantation, the patient is Mature cells of the cartilage extract trypsinization and centrifugation from the culture medium and transferred into transplanters environment containing 1.25 ml of sulphate gentamicin (concentration 70 µmol/l), 2.0 ml amphotericin b (trademark Fungizone®, antifungal drug, supplied from Suibb) - concentration of 2.2 mmol/l; 7.5 ml of 1-ascorbic acid (300 µmol/l), 25 ml of autologous serum (final concentration 10%) and the rest is DMEM/F12 to obtain 300 ml of the migration environment.

Then the support matrix 22 is cut to size, placing the bottom holes in the plate for culturing cells NUNCLON™, then placed in aseptic conditions on the bottom of the wells with 1-2 ml of the migration environment. A significant amount of cultivated prior to the maturation of cartilage cells (3-10 million Mature cartilage cells) in approximately 5-10 ml of the migration environment seeps into the support matrix 22 and incubated for approximately 72 hours at 37°C and 5% CO₂for further growth of cartilage cells.

During incubation of Mature cartilage cells are arranged in clusters and are bonded to the support matrix 22.

When using this method it was found that the support matrix 22 supports the growth and captures a significant number of Mature cartilage cells therein for forming implantable segment 20 without significant loss of biomechanical properties of the support matrix 22.

In addition, the support matrix 22 creates an environment to support continued growth of Mature cells of the cartilage after implantation of the implantable segment of the defective area of the cartilage.

In other the om embodiment, the Mature cells of the cartilage, after an incubation period 17-21 hours and determine the number of cells and their viability as described above, is placed in the transporting medium and grown directly on a support matrix 22 for at least 2 weeks.

It was found that implantable segment 20 may be temporarily deformed without mechanical damage or loss of Mature cells of the cartilage attached to the support matrix 22. This deformation is fully restored when entering implantable segment 20 into the joint or placing it on the surface intended for treatment, as described below.

Accordingly, in another embodiment of this invention the support matrix on which cells are grown, or just load it in large numbers, may be temporarily deformed to enable its introduction into a working instrument for the introduction of the implantable segment without mechanical damage or losses accrued cartilage cells.

It was also found that this matrix can be glued or attached mechanically to the defective area of the cartilage, without interfering with the further differentiation of Mature cells of the cartilage in their location and regeneration of natural substances matrix of cartilage.

In other variants, the invention includes tools for placement of implantable segment 0 in the site of implantation and mechanical locking device, holding implantable segment 20 at the site of implantation.

According to the present invention, the implant surgery is performed under arthroscopic technique. Figure 3 shows how the implantable segment 20 can be braided in diameter in the form of a spiral in the form of a cylinder to transplant such a way as to deliver the implantable segment to the implantation site through the working channel 26 arthroscopic conductor 28. The appropriate tool for the introduction of the implantable segment depicted in figure 4.

Figure 4 tool for the introduction of the implantable segment 30 includes a working channel 32 with a diameter and length that is convenient for input intended for the treatment of joint and delivery of implantable segment 20 of the required size.

For example, for most operations corresponding to the diameter of the working channel is in the range of about 8-20 mm, and its length is approximately 30-60 see Inside the working channel 32 with the possibility of longitudinal movement thereon is injection channel 34 located therein retractable and removable needle 36. The injection channel 34 is attached to the handle 38 which is telescopically at least partially inside the working channel 32. The needle 36 extends beyond the length of the injection channel 34 and allows the liquids to flow through it to the site of implantation. Injection the initial channel 34 is moved within the working channel 32 through telescopically movable handle 38 toward the site of implantation and from him.

Tool for the introduction of the implantable segment 30 further includes a cap 40 of rubber or other suitable material, attached to the instrument for the introduction of the implantable segment 30 is slidable. In working condition, cap 40 covers the defective area of the cartilage and keeps it liquid, such as blood and other natural fluids. In addition, the instrument for the introduction of the implantable segment 30 has two or more spaced move outside of the grips 42, holding the inner part of the working channel 32 can be moved toward each other, entering into gear (by moving the handle 38 to the destination), and reteplase from each other (by removal of the handle 38 from the destination). Such telescopic movement can be adjusted by the element offset (not shown)located in the handle 38, which allows the injection channel 34 and the hooks 42 to slide forward and backward within the working channel 32.

Figure 5-7 shows the normal arthroscopic surgery for implantation of the implantable segment 22 at the site of implantation, for example, the knee joint 10. The defective section 18 of the cartilage is removed to a depth of mostly above the subchondral layer 44, leaving a hole 46 (see Fig.6, 7). After removal of the defective area 18 of the cartilage defect area to prepare imposed the Yu implantable segment 22. If the subchondral layer was bleeding at the implantation site, this site cover any absorbent material which acts as a styptic.

In another embodiment, the preparation of the defective area may include performing injection of biocompatible glue through the needle 36 into the hole 46. Such biocompatible adhesive, indicated by the numeral 48 figure 6, may include fibrin glue (e.g., Tisseel®, based on fibrin glue, Baxter, Austria or fibrin glue is made in the operating room with the use of autologous blood samples).

Implantable segment 20 pre-cut to size and rolled into a spiral, forming a cylindrical shape, as shown in figure 5, and then hook the hooks 42 and hold the end of the tool for insertion of implantable segment 30. Supported on the end of the tool for insertion of implantable segment 30 of the implantable segment 20 is moved forward along the flow channel 33 to the site of implantation, unhooks the grips 42 and unwound with them or arbitrarily, leaving the working channel 32. A passage 33 includes one or more channels that have the possibility of putting tools in the area of transplantation, such as the instrument for the introduction of the implantable segment 30 and visualization tools.

Using Akhmatov 42 implantable segment 20 have thus, to the rough side 23 of the implantable segment was converted to the hole and gently held in place of the hole 46 to allow solidification of the adhesive 48 and gluing implantable segment to the hole 46.

In another embodiment of the inventions (7) to attach the implantable segment 20 in the hole 46 using a mechanical fastening device, for example, absorbable mechanical pins, clamps, screws or suture materials. Suitable for these purposes, the pins 50 are Ortho-Pin™, (commercially available pins of lactides copolymer, Ed. Geistlich Soehne, Switzerland).

On Fig shows one of embodiments of absorbable pin 50. In this embodiment, the pin 50 includes a head 52, an intramedullary canal 54 inside the barrel 56 and one or more retaining rings 58. The dimensions of the pin 50 will vary depending on the specific use, but usually the pin 50 has a length approximately in the range of 10-15 mm, the diameter of the head 52 is approximately 4 mm, the diameter of the intramedullary canal 54 approximately 1.2 mm, the diameter is approximately 2 mm, the diameter of the retaining rings is approximately 2.5 mm Retaining rings are used for fixation of the pin in the healthy section of the cartilage surrounding the defective area.

The pin 50 is made of any material that is harmless to the body and can be absorbed or otherwise is to advertise decay in the body after a period of time. For example, the pin 50 may be made of polylactide.

In the scope of the present invention also includes the use of a combination of adhesive 48 and mechanical fixation devices such as pins 50 for fastening the implantable segment 20 in the hole 46.

As shown in Fig.6, the second lock channel having one or two channels that can be used to skip them tools to the site of implantation for the auxiliary manipulation at the location of the implanted segment, adhesive and/or the placement of mechanical locking devices, or to access visualization tools to the site of implantation. A separate bushing channels can be used to perform one or more functions described in relation to an instrument for the introduction of the implantable segment 30 or other arthroscopic instrument.

As described above, when a defective area 18 distributed in cartilage subchondral layer 44 or requires the removal of the cartilage tissue at and below the subchondral region 44, as shown in figures 9 and 10, the above procedure is modified and includes the establishment of a hemostatic barrier 62 in the hole 46 to placing an implantable segment 20. Hemostatic barrier inhibits the growth or invasion of vascular tissue, osteocytes, fibroblast, etc. in otrabatyvaem joint. It is believed that it will not hinder the growth of tissue hyaline cartilage at the site of transplantation. Appropriate hemostatic barriers will inhibit the vascularization and invasion of cells in the developing cartilage to optimize the formation of cartilage and to achieve full recovery of the cartilage defect site.

Mainly hemostatic barriers sustained over a long period of time to fully restore cartilage and then they are absorbed or disintegrate in the body. Suitable hemostatic barrier is Surgicel® W1912 (Ethicon, Ltd., United Kingdom), absorbable hemostatic agent from oxidized regenerated sterile cellulose.

The above-described surgical instruments made from any material, for example metal and/or plastic or silicone, suitable for the manufacture of surgical instruments disposable or reusable.

Certain objects of the invention are illustrated in the experience of in vitro to study the behavior of Mature cells of cartilage tissue in contact with various support matrices. This experience in vitro suggests to investigate the ability of certain materials to withstand mechanical arthroscopic procedures, as well as the ability to obtain information about the growth of cartilage cells.

This is the invention can be illustrated by the following examples, which explain, but do not limit the scope of the present invention.

Example 1

Mature cartilage cells were grown for three weeks in a nutrient medium described above, in the incubator with CO₂at 37°With and manipulated in the laboratory class 100 in Verigen Transplantation Service ApS, Copenhagen, DK, or at the University of lübeck in Germany. It should be noted that other composition of the nutrient medium can also be used for culturing cells. The cells were fed with trypsin with trypsin add within 5-10 minutes and counted using staining for detection of viable cells of the dye Trypan Blue in the camera Burker-Turk (Burker-Turk). The number of cells is brought to 7.5×10⁵Mature cartilaginous cells per milliliter. One Cup NUNCLON™ was not covered in the laboratory class 100.

The material of the support matrix, in particular membrane collagen Chondro-Gide®, cut to size to attach to the bottom of the hole in the tablet NUNCLON™ for cell cultivation. In this case, the bottom of the tablet had a ring with a diameter of 4 cm under aseptic conditions.

After three weeks of Mature cells of the cartilage tissue was transferred from the culture medium in the transporting medium, described above, and approximately 5 x 10⁶Mature cartilaginous cells in 5 ml medium transfer was placed directly on the supporting matrix and dissipated on its surface the displacement. Cup incubated in an incubator with CO₂at 37°C for 3 days. At the end of this period, the Mature cells of the cartilage tissue was placed in clusters and were grown on a support matrix without removing them from the support matrix when washing their environment or with weak mechanical clicking on the matrix.

At the end of the incubation period the medium transfer decantation and the support matrix with the information contained therein is Mature cells of the cartilage tissue was cooled in 2.5%glutaraldehyde containing 0.1 M sodium salt of cakaudrove acid added as preservative. The support matrix were stained with Safranin O for histological analysis.

On figa shown black-and-white copy of a color micrograph.

On figa shown color micrograph for a better illustration of the features of the micrograph.

Example 2

Mature cartilage cells were grown for three weeks in a nutrient medium described above, in the incubator with CO₂at 37°and worked with them in the lab class 100 in Verigen Transplantation Service ApS, Copenhagen, DK or University glubka in Germany. The cells were fed with trypsin with trypsin EDTA for 5-10 minutes and counted by staining with the dye Trypan Blue in the camera Burker-Turk (Burker-Turk) to identify viable cells. The number of cells was adjusted to 5×10⁵of Mature cells is Radivoj tissue per milliliter. One Cup NUNCLON™ was not covered in the laboratory class 100.

The material of the support matrix, specified in example 1 was cut to a proper size for fixing to the bottom of the hole in the tablet NUNCLON™ for cell cultivation. In this case, on the bottom of the wells in the plate had a ring with a diameter of 4 cm under aseptic conditions.

After three weeks of Mature cells of the cartilage tissue was transferred from the culture medium in the transporting medium, described above, and about 5×10⁵Mature cartilaginous cells in 5 ml medium transfer was placed directly on the supporting matrix and dissipated on its surface. Cup incubated in an incubator with CO₂at 37°C for 3 weeks.

At the end of the incubation period the medium transfer decantation and the support matrix with the information contained therein is Mature cells of the cartilage tissue was cooled in 2.5% glutaraldehyde containing 0.1 M sodium salt of cakaudrove acid added as preservative. The support matrix were stained with Safranin O for histological development. For immunohistochemistry of collagen membranes were fixed in methanol-acetone and stained paint for aggrecan and collagen type II, using collagen rabbit type II, aggrecan type II mouse. Primary antibodies can be differentiated using fluorescent secondary antibodies.

On FIGU shown in black and white to the Pius color micrograph image of Mature cells of the cartilage 24.

On figw shown color copy micrograph for a better illustration of the properties of the micrograph.

Within three weeks of the incubation period of the reference matrix of Chondro-Gide® was observed the growth and reproduction of cartilage cells, with the formation of clusters in the center of the carrier and placing them in a line on the surface.

Example 3

Mature cartilage cells were grown for three weeks in a nutrient medium described above, in the incubator with CO₂at 37°and worked with them in the lab class 100 in Verigen Transplantation Service ApS, Copenhagen, DK or University glubka in Germany. The cells were fed with trypsin with trypsin etc for 5-10 minutes and counted using staining for detection of viable cells of the dye Trypan Blue in the camera Burker-Turk (Burker-Turk). The number of cells was adjusted to 5×10⁵Mature cartilaginous cells per milliliter. One Cup NUNCLON™ was not covered in the laboratory class 100.

The material of the support matrix, specified in example 1 was cut to a proper size for fixing to the bottom of the hole in the tablet NUNCLON™ for cell cultivation. In this case, the bottom of the tablet had a ring with a diameter of 4 cm under aseptic conditions.

After three weeks of Mature cells of the cartilage tissue was transferred from the culture medium in the transporting medium, described above, and about 5×10^{6/sup> Mature cartilaginous cells in 5 ml medium transfer was placed directly on the supporting matrix and dissipated on its surface. Cup incubated in an incubator with CO₂at 37°C for 3 weeks.}

^{The support matrix with the information contained therein is Mature cartilaginous cells were incubated with collagenase for 16 hours and then centrifuged. Cells were sown on a Cup NUNCLON™ and believed aliquots using viability staining Trypan Blue in the camera Burker-Turk (Burker-Turk).}

^{On figs shown their micrograph. The total number of counted cells was 6×106and the viability of more than 95%.}

Example 4

Experiments with animals were carried out in equipment of the University glubka, Germany.

In the knee joints of two sheep had been four defect with a diameter of 7 mm All intervention i. v. made under General anaesthesia Ketanest/Romana (Ketanest/Rompun). The defects produced by drilling two holes in the cartilage in weight bearing areas of the medial femoral condyle and the two holes in the plot femoral-nadkolennika and femoro-tibial joints.

Two defective parts one of each pair of holes passes through labels rise and fall of cartilage and subchondral layer above the bone, and another hole in each plot does not pass through these areas.

At the same time-piece the EC cartilage was removed from the area of the knees of sheep, not carrying the weight of the load.

Cells of the cartilage from the joint were grown to maturity on the support matrix according to example 3 in the next six weeks.

Mature cartilage cells, deposited on a support matrix, then implanted according to the method of arthroscopic surgery. One sheep was produced fixation using a bonding matrix to the defective area fibrin glue, and the other sheep matrix recorded polylactide pins, as described above in the present invention.

Sheep insulated from each other and the knee was fixed with a bandage for one week.

After that sheep were released for free circulation. The examination of the joint showed the removal of the defect attaching implanted reference matrix with defective cells to the site of cartilage and cartilage repair in the defective area.

In addition to the above-described method of growing Mature cartilaginous cells on a support matrix in a glass container, such as Cup NUNCLON™and changes in the nutritional environment and migration environment, as required for the proper cultivation of cells, the present invention also includes a method of growing Mature cartilaginous cells on a support matrix in a bioreactor, such as model No. 1302 supplied MinuCells GMBH Ltd., D-93077 BadAbbach, Germany.

Using the reactor, continuous flow pittel the Noah environment and migration environment is passed through the support matrix, when this cartilage cells can grow on a support matrix with greater speed without replacing the culture medium on the migration environment every another 24-96 hours, as required for a similar process using cups NUNCLON™.

The use of bioreactor causes the development of cartilage cells at an angle by passing a flow of nutrient medium and migration environment through the bioreactor.

On fig.11D shows the micrograph of Mature cartilage cells grown on a support matrix in a bioreactor.

The cultivation of Mature cells of the cartilage or in a glass bowl or on a support matrix can be carried out in a nutrient medium or in the medium of transfer to the complete maturation of cells. This means that no transfer of cartilage cells from the culture medium in the transporting medium. Mature cells of the cartilage tissue can be transferred from the culture medium in the transporting medium and Vice versa at any time of cultivation depending on the state of the cells, the stage of growth of cartilage cells and/or the patient's condition. Mature cells, cartilage cells or in the medium or in the migration environment, it is necessary to impregnate the support matrix for about 2-3 hours before transplantation to commit a significant amount of cartilage cells on it.

If the bioreactor is not used, the nutrient medium and seguiremos, used at a certain stage of cultivation, must be changed every another 24-96 hours depending on the number and viability of cells.

This invention includes the segment (and its use), consisting of a support matrix, predominantly elastic and absorbed into the body of a living organism, while supporting the matrix serves to support viable cells, fixed and grown on it for a certain minimum period of time. This fixation when grown cells can be penetrating into the surface of the matrix.

The support matrix is the integral physical structure together with a segment for ease of manipulation, for example, for implantation in the human body.

The dependent claims disclose how the objects of the present invention described above, and similar and equivalent embodiments of the known to the person skilled in the art, without going beyond the scope of this invention.

1. A method of treating defective area of the cartilage in animals by creating implantable segment, including Mature cells of the cartilage that is attached to the support matrix made of the possibility of absorption into the body of the specified animal transplantation implantable segment in the defective area of the cartilage and fixation of the implantable segm the NTA on the defective area of the cartilage.

2. The method according to claim 1, characterized in that before the fixation of the implantable segment remove defective cartilage tissue with the defective area of the cartilage, leaving the cartilage tissue in sufficient quantity to prevent bleeding from the cartilage in his defective area.

3. The method according to claim 1, characterized in that before the fixation of the implantable segment additionally set hemostatic barrier in the defective area of the cartilage.

4. The method according to claim 1, wherein the support matrix is made in the form of a sheet capable of supporting the growth of cartilage cells and create the physical integrity to the implanted segment.

5. The method according to claim 1, characterized in that the supporting matrix further includes polypeptides or proteins.

6. The method according to claim 1, characterized in that the supporting matrix made of collagen.

7. The method according to claim 6, characterized in that the collagen is selected from the group comprising mostly equine, porcine, bovine, sheep and chicken collagen.

8. The method according to claim 6, characterized in that use equine collagen.

9. The method according to claim 6, characterized in that the use of collagen type I.

10. The method according to claim 6, characterized in that the use of collagen type II.

11. The method according to claim 1, wherein the support matrix is made from a solid substance.

12. The method according to claim 1, characterized in that the supporting m the Trix perform gel.

13. The method according to claim 1, wherein the implantable segment is fixed on a defective area of the cartilage with glue.

14. The method according to item 13, wherein when the fixation choose fibrin glue.

15. The method according to claim 1, characterized in that during the commit phase of the implantable segment attached to the defective area mechanical locking devices with the possibility of absorption.

16. The method according to item 15, wherein as a mechanical fixing devices using a pin.

17. The method according to claim 1, characterized in that as the animal chosen people.

18. Tool for the introduction of the implantable segment containing Mature cells of the cartilage located on the support matrix, in the defective area of the cartilage of the animal containing an outer tubular shell having an end made to be maintained by the user, and the end made for introduction into the defective area of the cartilage, the handle and the telescopic element, located at the end of the shell, made for holding by a user, the injection channel, partially located in the handle and extending from the end of the shell, is made to be maintained by the user in the direction of the end shell made for introduction into the defective area of the cartilage, and hooks attached to the telescopic element and adaptirovan the e for engagement and release of the implanted segment during telescopic movement of the handle in the shell.

19. The tool p, characterized in that it further comprises a cap attached to the end of the shell, is made to be maintained by the user with the ability to slide and ejecting the fluid from the defective area of the cartilage.

20. Implantable segment to restore the cartilage of the animal by implantation, comprising a support matrix and Mature cells of the cartilage, fixed it, and the support matrix is made with the possibility of absorption into the body of the animal.

21. Implantable segment according to claim 20, wherein the support matrix is made in the form of a sheet capable of supporting the growth of cartilage cells and to physically create a seamless design with implantable segment.

22. Implantable segment according to claim 20, characterized in that the supporting matrix contains polypeptides or proteins.

23. Implantable segment according to claim 20, characterized in that the supporting matrix made of collagen.

24. Implantable segment according to item 23, wherein the collagen is selected from the group comprising mainly equine, porcine, bovine, sheep and chicken collagen.

25. Implantable segment according to item 23, characterized in that the collagen is selected porcine collagen.

26. Implantable segment according to claim 20, wherein the support matrix is made of a solid substance.

27. The implantable segme the t according to claim 20, wherein the support matrix is made gel.

28. Implantable segment according to item 23, characterized in that the collagen used collagen type I.

29. Implantable segment according to item 23, characterized in that the collagen used collagen type II.

30. Implantable segment according to claim 20, characterized in that it is made with the possibility of elastic deformation of the matrix.

31. Implantable segment according to claim 20, characterized in that the support matrix has a rough side.

32. Implantable segment according to claim 20, characterized in that the rough side of the support matrix is made porous.

33. Implantable segment according to claim 20, characterized in that the support matrix has a smooth side.

34. Implantable segment according to claim 20, characterized in that the support matrix has a rough and a smooth side.

35. A method of manufacturing an implantable segment containing Mature cells of cartilage tissue, fixed in the support matrix, including removing cartilage cells from the patient, culturing the Mature cells of cartilage tissue in a nutrient medium, the formation of the support matrix containing solid or gel-like element with the ability to maintain the growth of cartilage cells therein, and adding cultured Mature cells of the cartilage in the support matrix with the possibility of the ability to continue the growth of cultured cartilage cells on a support matrix and fixing them on him.

36. The method according to p, wherein the support matrix is made in the form of a sheet segment.

37. The method according to p, wherein the support matrix comprises polypeptides or proteins.

38. The method according to p, characterized in that the supporting matrix made of collagen.

39. The method according to § 38, wherein the collagen is selected from the group comprising mostly equine, porcine, bovine, sheep and chicken collagen.

40. The method according to § 38, characterized in that use equine collagen.

41. The method according to § 38, characterized in that the use of collagen type I.

42. The method according to § 38, characterized in that the use of collagen type II.

45. The method according to p, wherein the support matrix is made from a solid substance.

44. The method according to p, characterized in that the supporting matrix perform gel.

45. The method according to p, characterized in that the supporting matrix made of absorbable.

46. Implantable segment containing the Mature cells of the cartilage located on the elastic support matrix and glued to it with the possibility of maintaining the growth of cartilage tissue and penetration into the surface of the support matrix, characterized in that as the growth of Mature cartilage cells attach to the support matrix.

47. Implantable segment according to item 46, wherein the support matrix technology is sponsored with the possibility of absorption into the body of the animal.