SK13892003A3 - Method for autologous transplantation - Google Patents

Abstract

The present invention describes various support matrices to whichcells can adhere and proliferate. Such support matrices are useful for implantation in a wound site to promote healing and regeneration of damaged tissue. The present further describes an article including a membrance having at least one layer having a porous surface and also including submucosal intestine tissue, and cells adhered to the layer. The present invention further describes that the cells adhered to the layer include chondrocyte cells.

Description

~ _{0 &} fr) rie (FCZ autologous

Technical field

The present invention relates to the transplantation of chondrocyte cells, bone and cartilage grafts, healing, joint treatment and the like. prevention of pathological arthritis. In particular, the present invention relates to novel methods and tools for chondrocyte cell transplantation and cartilage regeneration.

BACKGROUND OF THE INVENTION

In the USA, more than 500,000 arthoplastic operations and total joint replacements are performed annually. Approximately the same number of similar operations will take place in Europe. This includes about 90,000 total knee replacement and around Praemer, S. Fumer, D. P.

50,000 knee defect operations (A.

Rice: Musculoskeletal Conditions in the United States, Park Ridge, III .: American Academy of Orthopedic Surgeons, 1992, 125). The most beneficial would be a method of regenerating cartilage treatment and could be done at an early stage of joint damage, thereby reducing the number of patients in need of artificial joint replacement surgery. Such preventive treatments would also reduce the number of patients with developed osteoarthritis.

The techniques used to smooth the surface of the cartilage structure in the joints mainly attempt to induce cartilage repair using subchondral drilling, abrasion and other methods by which the diseased cartilage and subchondral bone are removed and the vascularized spongy bone is exposed (J. Insall: Clin. Orhc. Clin. 1974, 101, 61

R. P. Ficat et al., Clin. Orthop. 1979, 144, 74; L. Johnson in: Operative Arthroscopy, (J. B. McGinty ed.) New York: Raven

Press, 1991, 341).

Coon and Cahn (Science, 1966, 153, 1116) have described a technique for culturing cartilage synthesizing cells from chicken embryonic primary cells. Later, Cahn and Lasher (PNAS USA, 1967, 58, 1131) used this system to analyze the participation of DNA synthesis as a prerequisite for cartilage differentiation. Chondrocytes respond to both EGF and FGF growth (Gospodarowicz and Mescher: J. Cell Physiology, 1977, 93,

117), but eventually lose their differentiation function (Benya et al. Cell, 1978, 15, 1313). Methods of chondrocyte growth have been described and are principally used with minor modifications as described by M. Brittberg et al. (M. Brittberg et al., New Engl. J. Med. 1994, 331, 889). Cells grown by these methods were used as autologous transplants into the knee joints of patients. Kolettas et al. moreover, they examined expression of cartilage-specific molecules such as collagens and proteoglycans in long-term cell culture. They have found that despite morphological changes during cultivation in single-layer cultures (A. Aulthouse et al .: Vitro Cell Dev. Biol., 1989, 25, 659; C. Archer et al .: J. Cell Sci., 1990, 97) , 361;

H. Haanselmann et al., J. Cell Sci., 1994, 107, 17; J.

Bonaventure et al., Exp. Cell Res., 1994, 212, 97), when compared to suspension cultures on agarose gels, alginate beds, or as spinner cultures (retaining spherical cell morphology), expressed markers such as type II and IX collagens and large aggregates proteoglycans, aggrecan, versican, and binding protein were not altered (E. Kolettas et al., J. Cell Science, 1995, 108, 1991).

Articular chondrocytes are specialized mesenchymal-derived cells found exclusively in the cartilage. Cartilage is an avascular tissue whose physical properties depend on the extracellular matrix produced by chondrocytes. During endochondral ossification, the chondrocytes mature, leading to cellular hypertrophy, characterized by the onset of collagen type X expression (WB Upholt and RR Olsen in: Cartilage Molecular Aspects (editors: B. Halí and Newman) CRC Boca Raton 1991, 43; E. Reichenberger et al. Dev: Biol., 1991, 148, 562, T. Kirsch et al .: Differentiation, 1992, 52, 89;

Stephens et al., J. Cell Sci., 1993, 103, 1111).

Despite advances in chondrocyte culture and bone and cartilage repair, there has been little success in attempting to transplant cartilage or chondrocytes to repair the joint surfaces. The teachings of the present invention provide factual and effective means for introducing a cartilage and / or chondrocyte transplant into a joint joint failure by which the cartilage is regenerated and the disorder corrected.

SUMMARY OF THE INVENTION

BRIEF SUMMARY OF THE INVENTION

It is an object of the present invention, in one embodiment, an implantable article, an implant comprising a support broth or bed that is capable of promoting cell growth and binding to the soil or bed, and a method of implanting such an implantable article to regenerate cells at the implant site. It is an object of the present invention, in one embodiment, a method of effectively treating the articular cartilage surface in an animal by transplanting an implant comprising chondrocyte cells retained in a supportive nutrient medium capable of being absorbed.

In one embodiment, the chondrocyte cells are retained only at the edge of the bed. In one embodiment, the support bed is a cover bed prepared from collagen and the chondrocyte cells are autologous or homologous. In another embodiment, the support broth is prepared from collagen and elastin or from collagen and one or more other resorptive materials. In another embodiment, the support culture medium is prepared from the small intestine submucosa of animal origin.

In another embodiment, the support bed is prepared from pericardium. In another embodiment, the support bed is prepared from collagen and one or more other polyester-based materials.

The implant is preferably fixed to the transplanted site by means of adhesive or mechanical fastening means. The present invention also provides a tool for locating and manipulating the implant at the implantation site and clips for securing the implant to the implantation site.

It is also an object of the present invention to provide an implantable cartilage repair product in animals comprising chondrocyte cells retained on a resorptive support medium and a method for producing the same.

In another embodiment, the present invention provides a method of treating the cartilage surface of an articular connection comprising placing the chondrocytes on the surface to be treated and covering the surface to be treated with a cover bed. The cover bed is attached to the cartilage area surrounding the failure.

DETAILED DESCRIPTION OF THE INVENTION

It is an object of the present invention, in one embodiment, to use certain products that inhibit the formation of vascular tissue, such as capillary capillaries protruding into the cartilage that has been formed during the process of autologous transplantation of chondrocytes into cartilage defects. Such products are beneficial in the repair of cartilage defects in the bones where the defects spread to or below the subchondral layer, sometimes referred to as defects across the entire thickness. The formation of vascular tissue from bone below the cartilage will tend to emerge into the new cartilage to be formed, leading to the appearance of cells other than the desired mesenchymal cells of specialized chondrocytes.

Contaminating cells introduced by vascularization can cause interference and excessive growth into the cartilage to be formed by implanted chondrocytes. One type of commercial product that can be used in the present invention is Surgicel® (Ethicon Ltd, UK), which is absorbed after 7-14 days. This is the opposite of the usual use of a hemostatic agent such as Surgicel®, as described in the enclosed product advertising by Ethicon Ltd.

We have surprisingly found that, in a situation where it is desired to suppress the re-vascularization of the cartilage, hemostatic material can be used, which will act as a gel-like artificial coagulate. Fig. 1C depicts a hemostatic T barrier that can be used to inhibit revascularization of a cartilage disorder. If red blood cells were present in an articular cartilage that is covered by such a hemostatic barrier in a defect over the entire thickness, these red blood cells would chemically turn into hematin and therefore will not be able to induce vascular growth.

The haemostatic product used as an inhibiting barrier for re-vascularization, such as Surgicel®, which contains or does not contain fibrin adhesives, is therefore effective for the envisaged method contemplated by the present invention. In another aspect of the invention, the nutrient broth, for example cell-free broth or other broth as described below, is used as a plaster covering a defective joint area or inserted into the area into which cultured chondrocytes are to be transplanted using, for example, autologous chondrocytes for transplantation. In the present invention, allogeneic or xenogeneic chondrocytes may further be used to heal the cartilage defect. Fig. 2 shows a support medium g which can be used as a patch to cover the defective area, and FIG. 3B shows the location of the cartilage defect 18.

Therefore, in one embodiment, the present invention provides methods for effectively repairing or treating cartilage defects on the surfaces of the articular bone connection, which comprises delivering an agent or composition to prevent vascular invasion at the cartilage site to be repaired, and also a culture medium, which isolates the repaired site and keeps the transplanted cells in the right place. The present invention also provides a kit comprising an optional hemostatic component for insertion into a site to be repaired, such that is an effective inhibition of vascularization to the site to be repaired; and once the chondrocytes to be transplanted are inserted into the site to be repaired, the nutrient medium 2 covers the repaired site so that the chondrocytes are held in place, but still have access to the nutrients.

Specific aspects of the invention have been exemplified in which an in vitro system has been used to study the behavior of chondrocytes in contact with a particular article or combination of certain articles that will inhibit the formation of vascular tissue. This in vitro testing predicts the ability of certain test materials to promote growth of the chondrocyte cell, tests the culture media for use as implants in which chondrocytes are retained, and tests the ability of each bed to inhibit vascularization as it happens in vivo, where capillary loops protruding into the cartilage arise during the process of autologous transplantation of the chondrocytes into the cartilage defect.

Suitable hemostatic preparations will be characterized by their ability to inhibit the growth or invasion of vascular tissue, osteocytes, fibroblasts, etc. into the emerging cartilage. A suitable haemostatic material fulfills the objectives of the methods of the present invention by preventing vascular and cellular invasion of the developing cartilage to optimize cartilage formation and achieve repair over the entire thickness of any articular cartilage disorders. Preferably, the hemostatic barrier will be stable for a sufficiently long time sufficient to allow for total cartilage repair and to be able to absorb or otherwise break down over time. One material found to have these properties is called Surgicel® W1912 (GG3DH Group, Ethicon Ltd. UK), and has hemostatic properties such as oxidized, regenerated, sterile cellulose.

The present invention also encompasses an implantable cartilage repair article, a method and an apparatus for such an implantable article. The implantable article comprises a support broth and autologous or homologous chondrocyte cells stored therein.

In one embodiment, the chondrocyte cells are retained only at one or more edges or layers of the feeder bed. Generally, the support culture medium is a material that will promote the growth of chondrocyte cells and which over time will be absorbed in the body of the patient receiving the implant. The transplantation can be performed by arthroscopic minimally invasive or open surgery techniques. The method of the invention also contemplates the use of suitable allogeneic and xenogeneic chondrocyte cells for repairing the cartilage disorder.

Such an implantable article is shown in FIG. More particularly, the implantable article 20 comprises a support nutrient medium 22 in which chondrocyte cells 24 are stored. A suitable support nutrient medium 22 is a solid or gel skeleton that characterizes the ability to maintain a stable shape at a time when it is allowed to grow therein. chondrocyte cells, both before and after transplantation, and which provides a system similar to the natural environment for chondrocyte cells to optimize chondrocyte cell growth differentiation.

The support broth 22 will be stable for a period of time sufficient to completely repair the cartilage and then absorb it over time, for example, over three months, without leaving any significant traces and without creating toxic degradation products. The term " absorb " is intended to include processes by which the support culture medium disintegrates by natural biological processes and the support culture medium disintegrates and its degradation products are eliminated, for example by lymphatic or blood vessels.

The support medium 22 is therefore preferably a non-antigenic membrane-like substance that is capable of being physiologically absorbed. Further, in one embodiment, the support broth 22 preferably has a film-like form having one relatively smooth side 21 and one relatively rough side 23. Rough side 23 faces cartilage disorder 18 and promotes growth of chondrocytes inwardly, while smooth side 21 is typically away from cartilage disorder 18 and prevents tissue ingrowth. In another embodiment, the support medium 22 has two smooth sides with similar porosity.

In one embodiment, the feeder support 22 is formed from polypeptides or proteins. The polypeptides or proteins are preferably obtained from natural sources, e.g. from mammals. However, artificial materials having comparable physical and chemical properties to polypeptides or proteins from natural sources can also be used to form the support broth 22. It is also preferred that the support nutrient soil 22 be reciprocally shaped as the user handles it so that the implant 20 can be handled and then returned to its original shape as described below in one aspect of the present invention.

The preferred material from which the support medium is formed is collagen, for example derived from horses, pigs, cattle, sheep or chickens.

Suitable materials from which the support broth 22 may be formed include ChondroCell® (commercially available collagen type II broth, Ed. Geistlich Sohne, Switzerland) and Chondro-Gide® (commercially available collagen type I broth, Ed. Geistlich Sohne) , Switzerland). The support broth 22 formed from type I collagen is somewhat stiffer than the support broth formed from type II collagen, although type II collagen broths may also be used. Another preferred material from which the support broth is formed is the cross-linked and non-cross-linked form of Permakol ™ (Tissue Science Laboratories, UK).

Otherwise, collagen is derived from marine sponge as described by Swatschek et al., Eur. J. of Pharmaceutics &

Biopharmaceutics, 2002, 53, 107-113 and in Australian patent application no. AU 3741701. In short, collagen is isolated from a marine sponge by washing the sponge several times with water, crushing and homogenizing the sponge tissue, treating the solution with high urea concentrations, centrifuging the resulting solution and precipitating the collagen from the supernatant.

For example, the implantable product described above can be prepared by culturing chondrocyte cells in this culture medium, as described in more detail below.

To prepare an autologous implant, the cartilage biopsy is first taken by an arthroscopic technique from the unloaded area of the patient's joint and transferred to a laboratory in a nutrient solution containing 20% fetal calf serum.

The cartilage biopsy is then mixed with an enzyme such as trypsinethylenediaminetetraacetic acid (EDTA), a proteolytic enzyme, and a binder to isolate and extract cartilage chondrocyte cells. The extracted chondrocyte cells are then cultured in a nutrient solution from an initial cell count of about 50,000 to a final number of about 20 million chondrocyte cells or more.

Three (3) days prior to re-implantation, the nutrient solution is changed to a transplant solution containing 10% autologous serum (i.e., serum extracted from the patient's blood as described below). The cultured chondrocyte cells in the transplant solution are allowed to soak up and penetrate into one or more layers of support broth 22 and continue to propagate to form implant 20. Preferably, the chondrocyte cells adhere to only one edge of the outer layer of support broth 22. The implant 20 is then implanted into the patient at the site of the cartilage disorder 18.

It will be understood that the disorder or injury 18 may be directly treated, slightly enlarged, or eliminated by the surgical procedure prior to implantation, as described in U.S. Patent Application Ser. The cultivation method as well as the nutrient and transplant solutions are described in detail below as exemplary embodiments of the invention, beginning with the description of the laboratory procedure for the harvested cartilage biopsy and the cultivation of chondrocyte cells, in accordance with the present invention. .

Culture solutions ("nutrient solutions") used to treat cartilage biopsies during the culture process and to grow cartilage chondrocyte cells are prepared by mixing 2.5 mL gentomycin sulfate (70 micromol / liter concentration), 4.0 mL amphotericin (concentration 2 Fungizone®, a fungicide available from Squibb), 15 mL of L-ascorbic acid (300 micromoles / liter), 100 mL of fetal calf serum (20% final concentration) and supplemented with DMEM / F13, to prepare 400 mL of nutrient solution. (The same nutrient solution is also used to transfer cartilage biopsy from the hospital to the laboratory where chondrocytes are extracted and multiplied.)

The blood obtained from the patient is centrifuged at approximately 3000 rpm to separate blood serum from other blood components. Separated blood serum is stored and used at a later stage of the culture process and in transplantation.

The cartilage biopsy previously obtained from the patient for autologous transplantation is sent in the nutrient solution described above to a laboratory where it will be cultured. In the laboratory, the cartilage biopsy is separated by decantation from the nutrient solution and discarded. The cartilage biopsy is then washed at least three times with pure DMEM / F12 to remove the fetal calf serum film from the cartilage biopsy surface.

The cartilage biopsy is then washed in a mixture containing the nutrient solution described above to which 28 mL of trypsin EDTA (concentration 0.06%) was added. The mixture is incubated for five to ten minutes at 37 ° C and 5% CO ₂ . After incubation, the cartilage biopsy is washed two to three times in the nutrient solution to wash the biopsy from the trypsin enzyme. The cartilage is then weighed. The minimum amount of cartilage typically required for chondrocyte growth is about 80-100 mg. More preferably, the amount is slightly greater, such as about 200 to 300 mg. After weighing, the cartilage is placed in a mixture of 2 mL of collagenase (which digests the enzyme; a concentration of 5000 enzyme units) with approximately 50 mL of pure DMEM / F12 solution, and crushed to partially digest the cartilage. After crushing, the cartilage is transferred through a funnel to a flask and about 50 mL of collagenase and a pure DMEM / F12 mixture are added. The crushed cartilage is then incubated for 17-21 hours at 37 ° C and 5% CO ₂ .

In one embodiment, the crushed incubated cartilage is then screened through a 40 µm sieve, centrifuged (at 1054 rpm, or 200 times g) for 10 minutes, and washed twice with nutrient solution. Then, the chondrocyte cells are counted to determine their ability to grow, after which the chondrocyte cells are incubated in the nutrient solution for at least two weeks at 37 ° C and in an atmosphere of 5% CO 2, during which time the nutrient solution is changed three to four times.

At least three days prior to re-implantation, the chondrocyte cells are removed from the nutrient solution by trypsinization and centrifugation and transferred to a transplant solution containing 1.25 mL gentamycin sulfate (70 micromol / liter concentration), 2.0 mL amphotericin (concentration 2, 2 micromoles / liter; Fungizone® trademark, fungicide available from Squibb), 7.5 mL of 1-ascorbic acid (300 micromoles / liter), 25 mL of autologous blood serum (10% final concentration) and supplemented with DMEM / F12 to prepare about 300 mL of transplant solution.

The support broth 22 is then trimmed to a suitable size for the well bottom of the NUNCLON ™ cell culture plate and then placed under aseptic conditions on the well bottom with 1-2 mL of transplant solution. A sufficient number of cultured cartilage chondrocyte cells (e.g., 3-10 million chondrocyte cells) in approximately 5-10 mL of transplant solution is then soaked into support broth 22 and incubated for approximately 72 hours at 37 ° C and 5% CO 2 in the cells. chondrocytes continue to grow. During this incubation, the chondrocyte cells are stacked in clumps and adhere to the support medium 22. In one embodiment, the chondrocyte cells adhere to only one outer layer of one side of the support medium 22. Using this method, the support medium 22 has been found to promote growth and growth. it retains in itself a sufficient number of chondrocyte cells to form the implant 20 without significantly losing the biomechanical properties of the supporting nutrient medium 22. The supporting nutrient medium 22 also provides an environment to promote continuous growth of the chndrocyte cells after implantation of the implant 20 at the cartilage defect site.

In another embodiment, after incubation for 17 to 21 hours, and after determining the number of cells and their viability, as discussed above, the chondrocyte cells are transferred to the transplant solution and then grown directly on the support broth 22 for at least two weeks as described above.

It has been found that the implant 20 can temporarily deform without mechanical damage or loss of chondrocyte cells that adhere to the support broth 22. This deformation is ultimately reversible once the implant 20 is inserted into the joint or placed on the surface to be treated as described below.

Accordingly, and in accordance with another aspect of the present invention, the support culture medium 22 on which the chondrocyte cells grow or have been introduced in sufficient numbers may temporarily deform in a manner that allows it to be introduced into the arthroscope working apparatus without being mechanically destroyed or without loss of chondrocyte cells deposited on it.

At the same time, it has been found that this support broth can be fixed to the cartilage defect site by adhesive or mechanical fastening means without compromising further in-situ differentiation of the chondrocytes and regenerating the natural cartilage bed material.

Other aspects of the present invention include tools for inserting the implant 20 at the implantation site and mechanical fastening means to maintain the implant 20 at the implantation site.

In one embodiment of the present invention, the implantation procedure is performed using arthroscopic techniques. Fig. 5 shows how the implant 20 can be rolled along its entire diameter to form a spiral cylindrical transplant such that the implant 20 can be delivered to the implantation site through the working channel 32 of the arthroscopic delivery device 30. A suitable arthroscopic delivery device is shown in FIG. 6th

The arthroscopic inserter 30 of FIG. 6 includes a working channel 22 'having a suitable length 35 and a diameter 31 for penetrating the joint and delivering the implant 20 with the desired dimension. For example, for most processes, the diameter 31 of the working channel 32 is about 8 to 20 mm and the length 35 is about 30 to 60 cm. Inside, there is a movable injection duct 34 with a withdrawal and replaceable needle 36, relative to the working duct 32. The injection duct 34 attaches to the handle 38, which is a telescopic withdrawal, at least in part, into the working duct 32. The needle 36 extends the length of the injection duct. 34 and allows fluids to be pushed through to the implantation site. The injection duct 34 is moved within the working duct 32 by means of a telescopically moving handle 38 to or from the implantation site.

The insertion device 30 also includes a cap, a bell 40 of rubber or other suitable material which is slidably applied to the insertion device 30. In use, the bell 40 surrounds the cartilage failure site and does not allow liquids such as blood and other natural fluids to flow into the failure site. cartilage. The delivery device 30 also has two or more outwardly deflected retaining members 42 attached to the handle 38 for holding, inserting, and inserting the implant 20 into the implantation site. As the handle 38 moves telescopically toward or away from the user, the retaining members 42 retract within the working duct 32 and move toward each other in a retaining manner (while the handle 38 moves toward the user) and release one from the other. grip (while the handle 38 moves away from the user). Such a telescopic movement can be controlled by an inclined element (not shown in the figure) located within the handle 38 that allows the injection channel 21 and the retaining element 42 to slide and retract within the working channel 32.

Figures 7 to 9 show a typical arthroscopic procedure for implantation of implant 22 at an implant site such as the knee joint 10. Defective cartilage 18 is removed from the site of the disorder, preferably to a depth above the subchondral layer 44, thereby forming a well 46 (see FIGS. 8 and 9). After removal of the defective cartilage 18, the site of failure is ready for insertion of the implant 22. If the subchondral layer is so disrupted that bleeding occurs at the site of implantation, the site may optionally be initially covered with any absorbent material that serves as a hemostatic barrier.

The site preparation may otherwise include injecting the live tissue compatible adhesive through the needle 36 into the well 46. Such a compatible living tissue adhesive, indicated in FIG. 8 as an adhesive 48 may comprise an organic fibrin adhesive (e.g., Tissel®, fibrin-based adhesive, Baxter, Austria, or a fibrin adhesive prepared in the operating room using an autologous blood sample).

The implant 20 is first cut to the desired size and coiled into a spiral-cylindrical shape as shown in FIG. 7, then engages in the retaining elements 42 and holds at the end of the arthroscopic delivery device 30. The arthroscopic delivery device 30 that holds the implant 20 at its end is then slid into the implantation site via the access channel 33, the implant is released from the holding elements 42 and unfolded by the retaining elements 42 or allowed to unfold as it exits the working duct 32. The access duct 33 includes one or more ducts 60 that allow the instruments, such as the inserter 22 and the observation device, to have access to the transplant site. Using the retaining elements 42, the implant 20 is manipulated such that the chondrocyte-bearing side, in this embodiment, the coarse side 23 of the implant 20, faces the well 46 and gently holds in place in the well 46 to allow adhesive 48 to solidify and hold implant 20 in well 46.

conduits, helpful

As shown in FIG. 7, a second access channel 60 having one or more can be used to allow instrument access to the site when implanting, adhesive, and / or mechanical fastening devices are deposited, or to allow observation devices to access the implant site. Such a separate access channel 60 may also be used to perform one or more of the functions described in relation to arthroscopic delivery device 30 or other arthroscopic instruments.

In another embodiment (Fig. 9), mechanical fasteners, such as clamps, hooks, screws or stitches that are capable of being absorbed, are used to secure the implant 20 in the well 46. Suitable terminals 50 include Ortho-Pin ™ (a commercially available lactide copolymer terminal, Ed. Geistlich Sohne, Switzerland). Fig. 10 depicts one embodiment of a clamp 50 that is capable of being absorbed. In this embodiment, the clamp 50 comprises a head 52, an intramedullary channel 54 within the axle 56, and one or more mounting rings 58. The dimensions of the clamp 50 will vary according to the particular application, but typically the clamp 50 is about 10-15 mm long, the head 52 has a diameter of about 4 mm, the intramedullary canal 54 has a diameter of about 1.2 mm, the axis 56 has a diameter of about 2 mm, and the fastening rings 58 have a diameter of about 2.5 mm. The fastening rings 58 serve to anchor the clip 50 in the healthy cartilage that surrounds the cartilage disorder. The clamp 50 is made of any material that will not be harmful to the body and is capable of absorbing after a certain period of time or otherwise being destroyed by the body. For example, the clamp 50 may be made of polylactide.

Also contemplated by the present invention is the use of a combination of an adhesive 48 and a mechanical fastener, such as a clamp 50, to secure the implant 20 into the well 46.

As indicated above, in cases where cartilage disorder 18 extends to or below subchondral layer 44, whether it requires cartilage removal to or below subchondral layer 44, as shown in FIG. 11 and FIG. 12, the above procedure may be modified to include insertion of hemostatic barrier 62 into well 46 prior to insertion of implant 20. In such defects, the physician may optionally use hemostatic barrier 62 to inhibit growth or invasion of vascular tissue, osteocytes, fibroblasts, etc. into the developing cartilage. This is believed to allow the formation of smooth cartilage at the transplant site. Suitable hemostatic barriers will inhibit vascularization and invasion of cells into the developing cartilage to optimize cartilage formation and to achieve cartilage growth across the thickness at the site of the disorder. The hemostatic barrier is preferably stable for a sufficiently long period of time to allow complete cartilage repair and after that time it is absorbed or otherwise degraded in the body. A suitable hemostatic barrier is Surgicel® W1912 (Ethicon, Ltd, United Kingdom), an absorbable hemostat formed from oxidized, regenerated, sterile cellulose.

The surgical instruments described above are made of any material, for example metal and / or plastic or silicone, suitable for the manufacture of single or multiple-use surgical instruments.

The support broth 22 or cover bed 2 of the present invention is formed from a collagen membrane as produced by the process described in US Pat. No. 5,028,695 (issued to Chemokol Gesellschaft zur Entwicklung von Kollagenprodukten), which is incorporated herein by reference. The collagen membrane can be prepared from raw beef or pork collagen material according to the following procedure: the collagen raw material is freed of fatty acid residues, washed with water, reacted in an alkaline medium, washed with water, washed with acid, washed with water, again is reacted with a strong alkali, then with an acid, causing swelling, reacted with an inorganic salt to shrink, water is extruded from the material to a dry weight of 40-50% by weight, the retained water in this material is removed by the addition of a solvent if necessary, the material can be crosslinked and then dried in a stretched form.

The support broth 22 or cover bed 2 may also be formed from a membrane comprising collagen, for example type I or type II collagen, and elastin, for example, from the membrane described in U.S. Pat. No. 5,397,353 (to Oliver et al., University of Dundee), which is incorporated herein by reference. Collagen / elastin membrane collagen can be obtained from equine, pork, bovine, sheep and chicken sources. In one embodiment, the support broth 22 or cover bed 2 is a collagen / elastin pig skin that has undergone a series of organic extractions to remove fat content therefrom. Once the fat has been removed, the sections are subjected to a series of enzyme extractions to remove all cellular material. Such a product, which is prepared in accordance with US Patent 5,397,353, is believed to be Permacol ™ and various cross-linked versions of Permacol ™.

Other membranes similar to Permacol ™ may be used in the present invention, for example Rapi-Seal Patch (Fusion Medical

Technologies, Inc., Fremont, CA) and the Tissue Repair Patch (Glycar

Vascular Inc., Dalias, TX).

The collagen / elastin membrane can be cross-linked up to 20% with polyisocyanates, preferably hexamethylene diisocyanate (HMDI). The collagen / elastin membrane used as a support broth 22 or as a cover bed 2 may be in the form of a foil having two smooth sides and pores of the same size and texture.

The collagen / elastin membrane preferably has the following specification: thickness 0.75 mm, length 4.8-5.2 cm, width 4.8-5.2 cm, collagen content greater than 79% and fat content 0.4%. Chondrocyte cells can be cultured on this bed to form an implantable article as described above. The implantable article may be inserted at or over the cartilage defect site.

In another embodiment, the support culture medium 22 can be made from Small Intestine Submucose (SIS). The method of preparing SIS from the small intestine segment is described in detail in U.S. Pat. No. 4,902,508, which is incorporated herein by reference. The intestinal segment, preferably a pig, sheep or bovine intestine, is first scraped by longitudinal wiping movements to remove both the outer layers (especially tunica serosa and tunica muscularis) and the inner layers (at least the luminal portions of tunica mucosa). The small intestine mucosa is typically dipped in brine and optionally stored in a hydrated or dehydrated state prior to use as described below. A plurality of superimposed layers of tissue of the small intestinal mucosa are then compressed, pressed against each other and shaped to provide a multi-layer remodeled structure as described in U.S. Pat. 5,788,625, 5,922,028 and 6,176,880 (all awarded

DePuy Orthopedics (Warshaw, IN)), which are incorporated herein by reference. The multilayer structure is provided with sufficient submucosal layers to provide a reconstructive tissue implant structure with a desired thickness to replace the endogenous cartilage structure.

Other SIS membranes that are useful in the present invention include Suspend Sling ™ from Mentor Corporation (Santa Barbara, CA), Staple Strips ™ from Glycar Vascular, Inc. (Dallas, TX), Surgical Fabrics from Boston Scientific (Natick, MA), SurgiSIS ™ Sling and SurgiSIS ™ Mesh from Cook Biotech, Inc. (West Lafayette, IN), SIS Hernia Repair Device by Sentron Medical, Inc. (Cincinnati, OH) and Restor® Soft Tissue Implant from DePuy Orthopedics.

Another membrane that can be used for the support broth 22 or cover bed 2 in accordance with the present invention is a collagen membrane which is capable of being resorbed and which is described in U.S. Pat. No. 5,837,278 (issued to Ed. Geistlich Sohne AG), which is incorporated herein by reference. This resorptive membrane can be obtained from naturally occurring membranes, for example, layers of skin with the face side, tendons and various animal membranes. This collagen membrane has a fibrous side in which cell growth is promoted and a smooth side that inhibits cell adhesion. The membrane is prepared by reaction with an alkali to saponify fats and degrade alkaline sensitive substances, by reaction with an acid to degrade acid sensitive substances, by washing, drying, defatting and, if necessary, crosslinking.

Other collagen membranes that may be used as a support nutrient 22 or cover bed 2 in accordance with the present invention include FortaFlex ™ (prepared from type I collagen) and GraftPatch® (prepared from cross-linked collagen) from Organogenesis, Inc. (Canton, MA). In addition, Equema Type I Antema® preparation from Opicrin S.p.A. is also useful in the present invention. (Corlo,

Italy).

Other membranes suitable for use as support broth 22 or cover bed 2 include a CollaTec membrane from Colla-Tec, Inc. (Plainsboro, NJ), Collagraft from NeuColl (Campbell, CA), BioMend from Integra Life Sciences Corporation (Plansboro, NJ), and BioMend® Absorbable Collagen Membrane from Collagen Matrix, Inc. (Franklin Lakes, N.J.). Biosynthetic Surgical Mesh from Advanced UroSciences, Inc., Brennen Medical, Inc. (St. Paul, MN), which is prepared from pork skin (essentially pure collagen) and BIOBAR ™ from Col-Bar, Ltd. (RamatHasharon, Israel) are also useful materials for support broth 22 or cover bed 2 in the present invention.

Moreover, collagen membranes with arranged fibers at the macromolecular level are furthermore also suitable for use as support broth 22 or as cover bed 2. Such membranes are described, for example, in the international patent publication no. WO 02/09790 to Mediolanum Pharmaceuticals S.P.A. and Opocrin S.P.A ..

In another embodiment, the support nutrient medium 22 and / or the cover bed 2 may be formed from collagen fibers which cross-link to each other through a reducing sugar or a derivative thereof, as disclosed, for example, in U.S. Pat. No. 5,955,438 (issued to ColBer R&D Ltd., Ramat-Hasharon, Israel), which is hereby incorporated by reference in its entirety. Such sugars may include, but are not limited to, ketones or aldehydes of monosaccharides, ribose, glycerose, threose, erythrose, lyxose, xylose, arabinose, allose, altrose, glucose, mannose, gulose, idose, galactose, diolose, talcose, diose, talcose, diolose trioses, tetrose, pentose, hexose, septose, octose, nanose or docose, and combinations of one or more of these carbohydrates. The support nutrient medium 22 and / or cover bed 2 may also include antimicrobials which have a therapeutic effect during cartilage regeneration. Such antimicrobial agents include penicillin, cephalosporins, tetracyclines, streptomycin, gentamicin, sulfonamides, fungicides such as myconazole, anti-inflammatory agents such as cortisone, and combinations of one or more thereof. Factors with tissue-inductive properties, such as fibroblast growth factor, platelet-derived growth factors, transformation growth factors, differentiation growth factors, and the like are also included in the support culture medium 22.

Other collagen materials useful in the invention are disclosed in U.S. Pat. 5,993,844 (granted to Organogenesis, Inc.), 6,206,931 (granted to Cook Biotech, Inc.) and 5,026,381 (granted to Colla-Tec Inc.), which are incorporated by reference and which may belong to one of the products discussed herein.

in submitted , 256,418 and no. US U.S. Patent No. 5,768,516; US U.S. Patent No. 5,768,516; the all here or more

Products not yet sold in the United States and which can be used as a support medium 22 or cover bed 2 include MACI-Maix® (Matricel, Herzogenrath, Germany), BioSeed C (Biotissue, Miami, FL) and VivesCart and PLA / PGA copolymers (IsoTis, Bilthoven, The Netherlands).

In another embodiment, the support broth 22 or cover bed 2 can be formed from porous collagen fiber material containing the antibacterial substances taurolidine and / or taurultam as described in EP 446,262 (granted by Geistlich Soehne AG, Wolhusen, Switzerland), which is herein incorporated by reference as link. The collagen porous mass can be obtained from commercial sources, for example from Pentapharm AG of Basel, Switzerland; Otto Suwelak Gmg Hof Billerbeck, Germany or from Ed. Geistlich Sohne A.G. Wolhusen, Switzerland. The collagen porous mass can also be produced by conventional methods. For example, the bovine skin may be treated chemically and mechanically to separate the epidermis from the lower associated fat. The layer can then be treated with moderately strong alkali, followed by acid and then washed. A proteolytic enzyme may be used to separate collagen from other proteins, and a lipase may be used to separate residual fat. The collagen product can then be reacted with an oxidizing agent, homogenized and lyophilized. The incorporation of taurolidine or taurultam may be accomplished prior to lyophilization or redissolution of lyophilized collagen in a solution of taurolidine or taurultam and re-lyophilization.

In another embodiment, the support broth 22 or cover bed 2 may be formed in accordance with the method for producing porous structures described in WO 99/27315 (Dip. Ing. Ingo Heshcel, Germany), which is incorporated herein by reference. The porous structures are formed from a liquid or paste mixture of substances which solidify at least partially by cooling the mixture between two submerged surfaces which can be tempered and have different temperatures. An ordered structure forms during solidification. The partially solidified product is then freeze-dried to give a homogeneous porous structure.

The support broth 22 or cover bed 2 may also be formed from one or more polymers that can be biologically absorbed, for example, collagen, fibrin, laminin and ibronectin, having large, interconnected pores, in accordance with the method described in the US patent no. No. 5,869,080 (issued to Johnson & Johnson Medical Inc., NJ), which is incorporated herein by reference. The bioabsorbable polymer may be cross-linked prior to freezing, for example with hexamethylene diisocyanate (HMDI).

The support broth 22 or cover bed 2 may be formed from a collagen porous mass such as Instat ™ (Johnson & Johnson) as described in Johnson & Johnson's brochure entitled Instat Collagen Absorbable Hemostat, September 1985, which is incorporated herein by reference.

In another embodiment, the support broth 22 or cover bed 2 can be made of a porous mass capable of being bio-absorbed in accordance with the method described in US Pat. 5, 700, 476 (Johnson & Johnson Medical Inc. _of NJ), which is incorporated herein by reference. The porous mass comprises a bed structure and at least one substructure and at least one pharmacologically active agent. The bed and substructure can be made, among others, from materials capable of bio-absorption, for example collagen, laminin, elastin and fibronectin. The porous bed may also contain one or more proteins or one or more polysaccharides or mixtures thereof. The pharmacological agent may include, inter alia, an antimicrobial, a cytokine, a growth factor, or an antibody.

In another embodiment, the support broth 22 or cover bed 2 is formed of collagen fibers as described in WO 96/25961 (Geistlich Soehne AG), which is incorporated herein by reference. The bed may further comprise a hydrogel-like substance which contains glycosaminoglycans, for example chondroitin sulfate, keratan sulfate, dermatan sulfate and hyaluronic acid and growth factors. An example of such a bed is described in U.S. Pat. No. 5,489,304 (Orgill et al.), Which is incorporated herein by reference in its entirety.

The support broth 22 or cover bed 2 may also be formed from a multi-layer membrane comprising a porous bed layer predominantly of collagen II and at least one dense barrier layer as described in WO 99/19005 (Geistlich Soehne AG) and US Patent Publication No. . 2002/0013627 (Geistlich et al.), Which are incorporated herein by reference. The bed layer has an open sponge texture and the barrier layer has a closed, relatively impermeable texture. The bedding layer may additionally contain glycosaminoglycans, including, but not limited to, hyaluronic acid, chondroitin sulfate, keratan sulfate, and dermatan sulfate. The bedding layer may also include laminin, fibronectin calcium alginate or anchorin II and growth factors. The barrier may be made of collagen I and III or synthetic materials, for example polyesters, homopolymers and copolymers of polyglycolic and polylactic acid, copolymers of glycolide and lactide, polyorthoesters and polycaprolactones.

Examples of membranes incorporating synthetic materials, such as polysters, are included in the present invention, and examples thereof are Paretex® Mesh and Parietex® Composite Mesh from Sofradim Production (Trevoux, France), SepraMesh ¹ * from Genzyme Corporation (Framingham MA) and Composix ™ Mesh from CR Bard, Inc. (Murray Hill, N.J.).

In a further embodiment of the invention, the support broth 22 and / or the cover bed 2 is formed from pericardium. Examples of membranes formed from pericardium that are useful in the present invention include Tutopatch® from Tutogen Medical, Inc. (Parsipanny, NJ), Peri-Guard membrane lines and Bio Vascular Sling from Bio Vascular (St. Paul, MN).

Certain aspects of the invention are exemplified by using an in vitro system to study the behavior of chondrocyte cells in contact with different carrier beds. These in vitro assays predict the ability of certain materials to withstand the arthroscopic procedure mechanically, and also provide information on the chondrocyte cell growth behavior.

These and other aspects of the present invention will be better understood from the following examples, by way of illustration and not limitation.

is possible of which it is

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. IA is a drawing depicting a typical articular end of bone 3. The bone material is typically coated on the surface of the joint with a cartilage cap 4. When the cartilage cap 18 is damaged or injured (cartilage cap well 18 in Fig. IB), the damaged site 18 can be treated directly or it can be slightly extended by surgical procedures. Optionally, a hemostatic barrier JL may be inserted into the damaged cartilage site to inhibit or prevent vascularization into the recovering cartilage from the underlying bone (Fig. 1C) when necessary, for example, disorders that extend to the subchondral layer or below it. The chondrocytes to be implanted into the cavity of the disorder are then layered on top of the hemostatic barrier 7 or directly on top of the lesion.

Fig. 2 is a drawing illustrating a disorder (gap in the dotted portion) in the cartilage cap (dotted portion) covered with a bed that is used to form a lid or patch or bandage (cover bed - 2) in a damaged area. This bed is fixed in place either by sewing or gluing to the edge of the cavity to a healthy cartilage or fixed differently.

The bed covers the damaged area of the joint into which cultured chorondrocytes have been transplanted.

Fig. 3A shows a typical articular end of the knee bone having a cartilage cap 16 on the articular surface.

Fig. 3B shows a cartilage defect or a cartilage cap injury to the articular end of bone 18.

Fig. 4 shows one embodiment of an implantable article (implant) 20 in accordance with the present invention.

Fig. 5 shows how the implant 20 of FIG. 4 for implantation by means of an arthroscopic delivery device 30, such as shown in FIG. 6th

Fig. 6 shows an arthroscopic delivery device 30 for implanting an implant 20 at an implant site 18 in accordance with the present invention.

Fig. 7 is a diagram schematically illustrating the insertion of the implantable article (implant) 20 of FIG. 5 to the site of the defect or injury 18 in the cartilage cap 16 by means of two access channels 33 and 60, which can accommodate arthroscopic instruments.

Fig. 8 is a schematic cross-sectional view of cartilage 16 with damage or injury that does not extend into the subchondral layer 44 and implant 20 in accordance with the present invention, which is fastened to the site of failure by adhesive 48.

Fig. 9 is a schematic cross-sectional view of cartilage 16 with damage or injury not extending into the subchondral layer 44 and implant 20 that is secured to the site of failure by mechanical fastening means 50.

Fig. 10 depicts one embodiment of a mechanical fastener 50 that is used to attach an implant to a defect or injury site.

Fig. 11 is a schematic cross-sectional view of cartilage 16 with damage or injury that extends into subchondral layer 44 and implant 20 in accordance with the present invention that is fastened to the site of failure by adhesive 48.

Fig. 12 is a schematic cross-sectional view of cartilage 16 with damage or injury that extends into the subchondral layer 44 and implant 20 that is secured to the failure site by mechanical fastening means 50.

Fig. 13A is a black and white copy of a color photomicrograph of a histological preparation at the beginning of chondrocyte cell growth on solid support broth.

Fig. 13AA is a digitized micrograph of FIG. 13A.

Fig. 13B is a black and white copy of the color photomicrograph of the histological preparation of the support broth of FIG. 13A with chondrocyte cells seeded after three weeks of growth on this solid support bed.

Fig. 13BB is a digitized micrograph of FIG. 13B.

Fig. 13C is a photograph showing a support medium formed from collagen in which chondrocyte cells grow, visualized by immunohistochemical staining.

Fig. 13D is a photograph showing a support medium formed from collagen in which chondrocyte cells grow in a bioreactor system, visualized by immunohistochemical staining.

Fig. 14 is a photomicrograph showing chondrocyte cells on DePuy support broth.

Fig. 15 is a graph depicting the total number of cells in the control (black fields), on the Chondro-Gide membrane (dotted fields) and on the DePuy group (gray fields) after three days, two weeks, and six weeks. For the indicated times, the values in the control sample are: 72,000, 575,167 and 528,333 cells. On the Chondro-Gide membrane:

109.167, 427.500 and 153.333. On DePuy membrane: 169.583,

494.167 and 100.833 cells.

Fig. 16 is a graph depicting cell viability (percentage) in control (black boxes), Chondro-Gide membrane (dotted boxes), and DePuy group (gray boxes) after three days, two weeks, and six weeks.

DETAILED DESCRIPTION OF THE INVENTION

EXAMPLE 1

Therefore, in order to use Surgicel® in accordance with our invention to prevent the development of blood vessels into autologically implanted cartilage or chondrocytes, we have allowed Surgicel® to react with a fixing agent, such as glutaraldehyde; we chose to react 0.6% glutaraldehyde with Surgicel® for one minute, followed by washing to remove glutaraldehyde residues that might otherwise be toxic to the tissue. Alternatively, Surgicel® was treated with a fibrin adhesive called Tisseel® (Immuno AG, Vienna, Austria) as described in Example 2 prior to reaction with glutaraldehyde. We found that

For example, Surgicel® fixed with a fixative such as glutaraldehyde, washed with sterile physiological saline (0.9%) and stored in a refrigerator does not dissolve for one to two months.

Surgicel® is generally resorbed between 7 and 14 days. This time would be too short because to prevent the development of blood vessels or vascularization as such from the bone structure to the implanted cartilage, a longer period of time is required before implanted chondrocytes injected into the solid cartilage layer obtain their necessary nutrition from the surrounding cartilage. In other words, a longer period of time, such as one month, is required to sufficiently inhibit vascularization. Therefore, the product should not be absorbed significantly before this time. On the other hand, resorption is finally necessary.

The organic material used as an inhibiting barrier must thus have these capabilities, and we have found that Surgicel® treated in this way provides this function.

EXAMPLE 2

Surgicel® was also coated with an organic adhesive and we used Tisseel® as the adhesive in this case. This product together with Surgicel® creates a useful barrier for our specific purpose. The Tisseel® was mixed as described below. Surgicel® was then coated with Tisseel® used as a spray on both sides of Surgicel® until wetted. Tisseel® (fibrin glue) was then allowed to solidify at room temperature. Immediately before complete solidification, Surgicel® was then placed in 0.6% giutaraldehyde for one minute and then washed with 0.9% sterile physiological saline. The pH was then adjusted with PBS and / or NaOH until it stabilized at 7.2-7.4. The thus treated Surgicel® was later washed in a tissue culture solution, for example, minimal stock / HAM F12 with 15 mM Hepes buffer.

As mentioned, in this example we used Tisseel® as a fibrin adhesive for wetting Surgicel®. Furthermore, the fibrin glue or adhesive can also be used directly at the bottom of the lesion towards the bone to which Surgicel® adheres. The in vitro system used, instead of in vivo testing, consists of a NUCLEON ™ Delta 6-well sterile dedicated cell research plate (NUNC (InterMed) Roskilde, Denmark). Each well is approximately 4 cm in diameter.

In the present invention, the fibrin adhesive agent may be any adhesive agent which together with the fibrin component provides an adhesive that can be accepted for human use (N. Ihara et al., Burns Ind. Therm. Inj., 1984, 10, 396). .

The invention also envisages any other adhesive component that may be used in place of the fibrin adhesive agent. In this example, Tisseel® or Tissucol® (Immuno AG, Vienna, Austria) was used here. The Tisseel® kit consists of the following components:

Tisseel®, a lyophilized, virally inactivated sealant containing protein coagulabilis, of which: fibrinogen, plasmafibronectin (CIG), factor coagulationis XIII, and plasminogen aprotinin (bovine) thrombin 4 (bovine) thrombin solution 500 (bovine chloride).

The Tisseel® kit contains the DUPLOJECT® application system.

The fibrin glue or two-component sealant using the Tisseel® kit is mixed as described in the Immune AG instructions.

EXAMPLE 3

Chondrocytes were cultured in a minimal stock culture solution containing HAM F12 and 15 mM Hepes buffer and 5 to 7.5% autologous serum in a CO ₂ incubator at 37 ° C and processed in Class 100 laboratory in Verigen Europe A / S, Symbion Science Park , Copenhagen, Denmark. For the cultivation of chondrocytes, another composition of the culture solution may be used. Cells were trypsinized with trypsin EDTA for 5 to 10 minutes and counted by trypan blue staining of viable cells in a Biirker-Turk chamber. The cell number was set to 7.5 x 10 ⁵ cells per mL. One NUNCLON ™ plate was not altered in the Class 100 laboratory.

Hemostatic distilled did not flush

The Surgicel® haemostatic barrier was trimmed to a suitable size that fits the bottom of the well in a NUNCLON ™ tissue culture plate. In this case, a circle of approximately 4 cm diameter (but could be of any size) and was placed under aseptic conditions on the bottom of a NUNCLON ™ tissue culture well. Delta 6-well Disposable Cell Research Plate (NUNC (InterMed) Roskilde, Denmark). The hemostatic barrier to be placed at the bottom of the well was pretreated as described in Example 1. This treatment significantly delays the absorption of Surgicel®.

the barrier was then washed several times with water and then several times until unreacted glutaraldehyde. A small amount of cell culture solution containing serum was used to absorb into the hemostatic barrier and keep the hemostatic barrier wet at the bottom of the well.

at the same time

Approximately 10 ^{s of} cells in 1 mL of culture solution were placed directly on top of the hemostatic barrier, spread on the surface of the hemostatic barrier, which was pretreated with 0.4% glutaraldehyde as described above. The plate was then incubated in a CO ₂ incubator at 37 ° C for 60 minutes. An amount of 2 to 5 mL of tissue culture solution containing 5 to 7.5% serum was carefully added to the well containing the cells by holding the pipette tip tangential to the wall of the well at the outlet of the solution so as not to rinse the cells with serum. The pH of the solution turned out to be too low (pH about 6.8). The pH was then adjusted to 7.4 to 7.5. The following day, some chondrocytes began to grow on the hemostatic barrier and formed clumps. Some cells died because they were exposed to low pH before treatment. The plate was incubated for 3 to 7 days with solution exchange on the third day.

At the end of the incubation time, the solution was decanted and the plate was discarded

cooling after the addition 2.5% glutaraldehyde containing 0,1 M dimethylarzinic acid sodium salt, also called sodium cacodylate, with a pH adjusted to 7.4 as HCl a fixative for the cell preparation and as a carrier (hemostatic barrier) for later preparation for electron microscopy. EXAMPLE 4 Chondrocytes are cultivated in minimum primary

culture solution containing HAM F12 and 15 mM Hepes buffer and 5 to 7.5% autologous serum in a CO ₂ incubator at 37 ° C and processed in Class 100 laboratory in Verigen Europe A / S, Symbion Science Park, Copenhagen, Denmark. For the cultivation of chondrocytes, another composition of the culture solution may be used. Cells were trypsinized with trypsin EDTA for 5-10 minutes and counted by trypan blue staining of viable cells in a Bürker-Túrk chamber. The cell number was set to 7.5 x 10 ⁵ cells in 1 mL. One NUNCLON ™ plate was not altered in the Class 100 laboratory.

Surgicel® (for use as a haemostatic barrier) was reacted for 1 minute with 0.6% glutaraldehyde as described in Example 1 and washed with 0.9% sterile sodium chloride solution or preferably with a buffer such as PBS buffer or culture solution, for example MEM / F12 since the pH after reaction with glutaraldehyde is 6.8 and preferably should be 7.0 to 7.5. On both sides of Surgicel®, Tisseel® was applied with the DUPLOJECT® system to cover both sides of Surgicel®, the intended patch, with fibrin glue. The adhesive is allowed to dry under aseptic conditions for at least 3 to 5 minutes. The coated hemostatic barrier was placed on the bottom of the well in a NUNCLON ™ Delta 6-well sterile disposable cell research plate (NUNC (InterMed) Roskilde, Denmark). A small amount of serum-containing tissue culture solution was used to be absorbed into the hemostatic barrier. Approximately 10 ⁶ cells in 1 mL of serum-containing tissue culture solution were placed directly on top of the hemostatic barrier dispersed on the hemostatic barrier surface. The plate was then incubated in a CO ₂ incubator at 37 ° C for 60 minutes. An amount of 2 to 5 mL of tissue culture solution containing 5 to 7.5% serum was carefully added to the well containing the cells by holding the pipette tip tangential to the wall of the well at the outlet of the solution so as not to rinse the cells with serum. After 3 to 6 days, microscopic examination showed that cells adhered to and grown to Surgicel® satisfactorily, suggesting that Surgicel® did not show toxicity to chondrocytes and that chondrocytes satisfactorily grew into Surgicel®.

The plate was incubated for 3 to 7 days with solution exchange on the third day. At the end of the incubation, the solution was decanted and the plate was chilled by the addition of 2.5% glutaraldehyde containing 0.1 M dimethylarzine acid sodium, also called sodium cacodylate, with a pH adjusted to 7.4 as HCl as a fixative for the cell preparation and as a carrier (haemostatic barrier) for later preparation for electron microscopy.

EXAMPLE 5

Chondrocytes were cultured in a minimal stock culture solution containing HAM F12 and 15 mM Hepes buffer and 5 to 7.5% autologous serum in a CO2 incubator at 37 ° C and processed in Class 100 laboratory in Verigen Europe A / S, Symbion Science Park, Copenhagen, Denmark. Cells were trypsinized with trypsin EDTA for 5-10 minutes and counted by trypan blue staining of viable cells in a Burker-Tiirk chamber. The cell number was set to 7.5 x 10 ⁵ to 2 x 10 ⁶ cells in 1 mL. One NUNCLON ™ plate was not altered in the Class 100 laboratory.

Bio-Gide® is a resorbing bilayer membrane that is used as a patch or bandage covering the area of a joint disorder into which cultured chondrocytes are to be transplanted by autologous transplantation. Bio-Gide® is a pure collagen membrane obtained by a standardized, controlled industrial process by E. D. Geistlich Sohne AG, CH-61 10 Wolhusen. Collagen is extracted from veterinary certified pigs and thoroughly cleaned to avoid antigenic reactions and sterilized by double gamaradiation. The bilayer membrane has a porous surface and a dense surface. The membrane is made of type I and type III collagen without further cross-linking or chemical treatment. Collagen is resorbed within 24 weeks. The membrane retains its own structural integrity even when wet and can be fixed by sutures or cloves. The membrane may also be "bonded with a fibrin glue, such as Tisseel®," to the surrounding cartilage or tissue, either in place of the stitch or together with the stitch.

Bio-Gide® was not altered in Class 100 laboratory and was placed under aseptic conditions on the bottom of NUNCLON ™ tissue culture 6-well Delta Sterile Disposable Cell Culture Plate (NUNC (InterMed) Roskilde, Denmark), either with a porous bilayer surface of the membrane facing upwards or with the dense surface of the membrane facing upwards. Approximately 10 ⁶ cells in 1 mL of serum-containing tissue culture solution were placed directly on top of Bio-Gide®, dispersed on either porous or dense Bio-Gide® surface. The plate was then incubated in a CO ₂ incubator at 37 ° C for 60 minutes. An amount of 2 to 5 mL of tissue culture solution containing 5 to 7.5% serum was carefully added to the well containing the cells by holding the pipette tip tangential to the wall of the well at the outlet of the solution so as not to rinse the cells with serum.

On the second day after the chondrocytes were placed in a well containing Bio-Gide®, the cells were examined with a Nikon Invert microscope. Some chondrocytes were found to adhere to the edge of Bio-Gide®. It was of course not possible to look through this microscope through Bio-Gide® alone.

The plate was incubated for 3 to 7 days with solution exchange on the third day. At the end of the incubation, the solution was decanted and the plate was chilled by the addition of 2.5% glutaraldehyde containing 0.1 M dimethylarznic acid sodium salt, also called sodium cacodylate, with a pH adjusted to 7.4 as HCl as a fixative for the cell preparation and as carrier for Bio38

Gide® with cells grown on either porous or dense surfaces. Bio-Gide® patches were then sent for electron microscopy at the Department of Pathology, Herlev Hospital, Denmark.

Electron microscopy showed that chondrocytes cultured on the dense surface of Bio-Gide® did not grow into the collagen structure of Bio-Gide® while cells grown on the porous surface grew into the collagen structure and further showed the presence of proteoglycans and no evidence of fibroblast structures. This result showed that when a collagen patch, such as a Bio-Gide® patch, is sewn as a patch covering the cartilage disorder, the porous surface of the collagen bed should be facing downwards towards the disorder into which the chondrocytes are to be injected. The chondrocytes will then be able to penetrate collagen and form a smooth cartilage surface in line with the intact surface, and a smooth layer of proteoglycans will form in this region. While the dense surface of the collagen patch faces down towards the disorder, the chondrocytes to be implanted will not associate with the collagen and the cells will not produce the same smooth surface as described above.

EXAMPLE 6

Chondrocytes were cultured in a minimal stock culture solution containing KAM F12 and 15 mM Hepes buffer and 5 to 7.5% autologous serum in a CO 2 incubator at 37 ° C and processed in Class 100 laboratory in Verigen Europe A / S, Symbion Science Park, Copenhagen, Denmark. Cells were trypsinized with trypsin EDTA for 5-10 minutes and counted by trypan blue staining of viable cells in a Burker-Turk chamber. The cell number was set to 7.5 x 10 ⁵ to 2 x 10 ⁶ cells in 1 mL. One NUNCLOPP 'plate was not altered in the Class 100 laboratory.

The Bio-Gide® membrane used as a resorptive bilayer membrane can also be used together with an organic adhesive, such as Tisseel®, with an additional significantly higher aprotinin content than is typically found in Tisseele®, as described in the advertised product. By increasing the aprotinin content to about 25,000 KIU / mL, the resorption of the material is delayed for weeks instead of the usual duration in days.

To test this feature in vitro, Tisseel® was applied to the bottom of a NUNCLON ™ well and allowed to solidify completely. A collagen patch, such as Bio-Gide®, was then applied to Tisseel® and adhered to the bottom of the well. This combination of BioGide® and Tisseel® is designed to form a haemostatic barrier that will inhibit or prevent the development or infiltration of blood vessels into the area of chondrocyte transplantation. This hybrid collagen patch can now be used both as a hemostatic barrier at the bottom of the disorder (closest to the surface to be repaired), but also as a support bed for cartilage formation because the distal surface may be the porous side of the collagen patch to promote chondrocyte infiltration and cartilage bed. Thus, this hybrid collagen patch can also be used to cover the top of the implant with a porous collagen surface facing the implanted chondrocytes and as a barrier forming top. A hybrid collagen patch with an increased aprotinin component can also be used without any organic glue, such as Tisseel®, and placed directly into a failure by adherence by natural forces. The collagen patch can therefore be used both as a hemostatic barrier as well as a cell-free covering of the repaired or transplanted site, with the porous surface of the patch oriented towards transplanted chondrocytes or cartilage. Another variant would employ a collagen patch that consists of type II collagen (Geistlich Sohne AG, CH-61 10 Wolhusen).

Accordingly, the present invention provides a hybrid collagen patch, wherein said patch is a collagen bed with elevated levels of the aprotinin component, preferably about 25,000 KIU / mL, in conjunction with an organic bed adhesive, wherein the collagen component is similar to a Bio-Gide® bilayer material capable of absorption or collagen type II and the organic adhesive is similar to Tisseel®. In another embodiment, no hybrid adhesive is used in the hybrid collagen patch to adhere to the repair site.

EXAMPLE 7

The kit, as foreseen, will allow suitable practice for the method of the present invention. In a preferred embodiment, the kit of the invention provides sterile components suitable for trouble free use in an operating environment and provides a suitable hemostatic barrier, a suitable patch, and if necessary a suitable organic adhesive. The kit of the invention may also provide sterile, cell-free bed material as a support for autologous chondrocytes to be implanted into a failure on the surface of the hinge. In one embodiment, the kit of the invention comprises a Surgicel® haemostatic barrier and a Bio-Gide® topsheet together with a suitable organic adhesive to coat the Tisseel® haemostatic barrier, wherein Surgicel® and Bio-Gide® have been treated in accordance with the teachings of the present invention for time extension, before resorption occurs. In cases where Tisseel® is pre-coated, in one embodiment, Tisseel® is supplemented with additional aprotinin to increase resorption time.

In another preferred embodiment, the hemostatic barrier and cover patch are of a semi-permeable collagen matrix that has been treated to increase the resorption time of the material. It is also possible to provide the Tisseel® adhesive in an increased amount as a special ingredient to be applied as needed, due to the inherent variability and uniqueness of the circumstances that any repair or transplant operation will encounter.

It will be appreciated by those skilled in the art that many variations and / or modifications of the invention may be made, as shown in particular embodiments, without departing from the spirit or scope of the invention as described in its entirety. The present embodiments and examples are therefore to be understood in all respects as illustrative and not restrictive.

EXAMPLE 8

Chondrocyte cells were cultured for three weeks in the above culture solution in a CO ₂ incubator at 37 ° C and processed in Class 100 laboratory at Verigen Transplatation Service A / S, Copenhagen, Denmark or the University of Lubeck, Germany. (Note that other mixtures of culture solutions may also be used to cultivate chondrocyte cells.) Cells were trypsinized with trypsin EDTA for 5 to 10 minutes and counted with trypan blue staining of viable cells in a BirkerTurk chamber. The cell number was set to 7.5 x 10 ^sec cells per milliliter. One NUNCLON ™ plate was not altered in the Class 100 laboratory.

The support broth material, namely the Chondro-Gide® collagen membrane (identical to Bio-Gide® except that Chondro-Gide® has a wider and longer than Bio-Gide®, both available from Ed Geistlich Sohne, Geistlich Pharma AG, Wolhusen, Switzerland) was trimmed to a suitable size to fit into the bottom of a well on a NUNCLON ™ cell culture plate. In this case, under aseptic conditions, a circle of about 4 cm diameter was placed at the bottom of the well.

After three weeks, the chondrocyte cells were transferred from the culture solution to the transplant solution described above, and about 5 x 10 ⁶ chondrocyte cells in 5 mL of the transplant solution were placed directly on top of the support broth and dispersed on its surface. The plate was incubated in a CO ₂ incubator at 37 ° C for three days. After this time, the chondrocyte cells were lined up and started to grow on the support broth and could not be removed from the support bed by rinsing it with a solution or even mechanically using a slight pressure on the bed.

At the end of the incubation, the transplant solution was decanted and the support broth with chondrocyte cells injected therein was frozen in 2.5% glutaraldehyde containing 0.1 M dimethylarzine acid sodium salt added as a fixative. The support broth was stained with safranin O for histological evaluation. A black and white copy of the color micrograph is shown in FIG. 13A. To better illustrate the features of the micrograph, its digitized form is also shown in FIG. 13AA.

EXAMPLE 9

Chondrocytes were cultured for three weeks in a culture solution described above in a CO ₂ incubator at 37 ° C and processed in Class 100 laboratory at Verigen Transplantation Service A / S, Copenhagen, Denmark, or the University of Ltlbeck, Germany. Cells were trypsinized with trypsin EDTA for 5-10 minutes and counted by trypan blue staining of viable cells in a Burker-Turk chamber. The number of chondrocyte cells was set to 5 x 10 ⁵ cells per milliliter. One NUNCLON ™ plate was not in the Class lab

100 changed.

Chodnro-Gide® support broth, as in Example 1, was trimmed to a suitable size to fit into the bottom of a well on a NUNCION ™ cell culture plate. In this case, under aseptic conditions, a circle of about 4 cm diameter was placed at the bottom of the well.

After three weeks, the chondrocyte cells were transferred from the culture solution to the transplant solution described above, and about 5 x 10 ⁵ chondrocyte cells in 5 ml transplant solution were placed directly on top of the support broth and dispersed on its surface. The plate was incubated in a CO 2 incubator at 37 ° C for three weeks.

At the end of the incubation, the transplant solution was decanted and the support broth with the chondrocyte cells growing therein was frozen in cold in 2.5% glutaraldehyde containing 0.1 M dimethylarzine acid sodium salt added as fixative. The support broth was stained with safranin O for histological evaluation. For immunohistochemistry, collagen membranes were fixed in methanol-acetone and stained for aggrecan and type II collagen using rabbit anti-human collagen type II and mouse anti-human aggrecan. Primary antibodies were visualized with fluorescent secondary antibodies. A black and white copy of the color micrograph in FIG. 13B shows chondrocyte cells 24. To better illustrate the features of the micrograph, its digitized form is also shown in FIG. 13bb.

During a three-week incubation on Chondro-Gide © support broth, chondrocyte cells were observed to grow and multiply on the nasal bed, forming clumps in the middle of the nasal bed and leveling along the surface.

EXAMPLE 10

Chondrocytes were cultured for three weeks in a culture solution described above in a CO ₂ incubator at 37 ° C and processed in Class 100 laboratory at Verigen Transplantation Service A / S, Copenhagen, Denmark, or at the University of Libya, Germany. Cells were trypsinized with trypsin EDTA for 5-10 minutes and counted by trypan blue staining of viable cells in a Bürker-Túrk chamber. The number of chondrocyte cells was set to 5 x 10 ⁵ cells per milliliter. One NUNCLON ™ plate was not altered in the Class 100 laboratory.

Chodnro-Gide® support broth, as in Example 1, was trimmed to a suitable size to fit into the bottom of a well on a NUNCLON ™ cell culture plate. In this case, under aseptic conditions, a circle of about 4 cm diameter was placed at the bottom of the well.

After three weeks, the chondrocyte cells were transferred from the culture solution to the transplant solution described above, and about 5 x 10 ⁵ chondrocyte cells in 5 ml transplant solution were placed directly on top of the support broth and dispersed on its surface. The plate was incubated for three weeks in a CO ₂ incubator at 37 ° C.

The support broth with cultured chondrocyte cells was then incubated for 16 hours. The support broth with chondrocyte cells was then centrifuged. Cells were seeded on a NUNCLON ™ plate and an aliquot was counted by staining viable cells with trypan blue in a BurkerTurk chamber. In FIG. 11c shows her photomicrograph.

The total calculated cell count was found to be 6 x 10 ⁶ and the viability was greater than 95%.

EXAMPLE 11

This example describes a membrane toxicity and biocompatibility assay prepared according to U.S. Pat. No. 4,902,508 (issued to DePuy, a subsidiary of Ethicon, Inc .; a DePuy membrane or "Ethicon") and a Chondro-Gide ® membrane (Ed Geistlich Sohne, Geistlich Pharma AG, Wolhusen, Switzerland). The viability and number of chondrocyte cells that adhered to DePuy and Chondro-Gide® membranes after three days, two and six weeks was determined by visual counting of the number of cells that adhered to the membranes.

The DePuy membrane was tested with a Chondro-Gide® membrane as a positive control and a negative control using the same method, but without any membrane.

Previously frozen human chondrocyte cells (14 million cells) were thawed, washed and cell number and viability determined. After thawing, 3.2 million cells were recovered at 87% viability. To each of the two tissue culture flasks was added 1.6 million cells at a concentration of 5.3 x 10 ⁴ cells per milliliter and incubated at 37 ° C for three days. The resulting cell number and viability in the flasks was 3.5 million cells with 98% viability, respectively. 3.6 million cells with 93% viability.

Cell number and viability were analyzed for six samples from each membrane and for six samples of the control group without membrane. Membranes were analyzed after three days, after two and six weeks. DePuy membrane samples

Chondro-Gide® was trimmed to a square size of one square inch (i.e. 6.4516 cm ² ). Due to the dry consistency of the DePuy membrane, it was somewhat more difficult to cut the membrane.

Chondrocytes were trypsinized and cell viability and cell counts were determined as indicated above. Cells were centrifuged and resuspended to a concentration of one million cells per milliliter.

DePuy and Chondro-Gide® membranes were washed twice with phosphate buffered saline (PBS) to pH 7.17. Each membrane was placed in a culture plate well. One hundred microliters of chondrocyte cell suspension at a concentration of one million cells per milliliter was applied to each membrane and to the bottom of six wells without membrane. Another culture solution (3 mL) was added to each well. The culture plates were incubated for at least three days at 37 ° C.

Fig. 14 illustrates chondrocyte cells that adhered to the DePuy membrane. Cells were harvested and counted after three days, two weeks, and six weeks.

DePuy and Chondro-Gide® membranes were treated with an enzyme solution containing a mixture of two milliliters of 0.25% trypsin and one milliliter of collagenase (5000 units in total) to dissolve the membranes and determine the number of cells and their viability. Once the membrane had dissolved, chondrocyte cells were harvested by centrifugation and counted. The treatment time of the enzyme solution varies depending on the type of membrane. For the DePuy membrane, the collagenase reaction took much longer than for the Chondro-Gide® membrane. The DePuy membrane did not completely dissolve after two hours, while the ORondride® Gide® membrane completely dissolved after about one to one and a half hours. Therefore, in order to avoid cell stress, digestion with collagenase should not last more than two hours.

The chondrocyte control group was trypsinized, washed and pelleted by centrifugation. The cells were then resuspended in DMEM solution to determine the final cell count.

The results of the experiment are given below. There were no significant differences in the viability of the control cells when compared to cells grown on DePuy and Chondro-Gide ® cots. The viability in the control group, the Chondro-Gide ® carrier, and the DePuy carrier were, as shown in FIG. 16, high, about 87% for the control group, about 94% for the Chondro-Gide® cot and 93% for the DePuy cot. After a two week interval, viability in the control group was about 90%, for the Chondro-Gide® group about 91% and about 83% for the DePuy group. After six weeks, about 80% of the cells were viable in the control group, while about 74% were viable on the Chondro-Gide membrane and about 74% were viable under DePuy carrier bed conditions. Thus, there is no wider range in cell viability under different growth conditions.

The specific number of cells was not significantly affected by the DePuy and Chondro-Gides® carrier bed until late cultivation. As shown in FIG. 15, after three days, the control group had about 72,000 cells, while the Chondro-Gide ® carrier was about 109,167 and the DePuy carrier was about 169,583 cells. After two weeks, the control cells multiplied about eight times to 575.167 cells, while the Chondro-Gide® cells nearly four times to number 427.500 and the DePuy cells nearly three times to 494.167. Finally, after six weeks, the number of cells in the control group decreased slightly to 528,333, the number of cells on Chondro-Gide® and DePuy dropped dramatically to 153,333, respectively. 100,833th

While this invention has been described with reference to characteristic embodiments, it will be understood that those skilled in the art may design other embodiments and variations of the invention without departing from the true spirit and scope of the invention. The appended claims are intended to include all such embodiments and equivalent variations thereof.

List of reference marks

- haemostatic barrier

- cover bed, patch

- bone

- cartilage cap 10 - knee joint

- thigh-bone

- tibia

- healthy cartilage

- defect, defect or injury of the cartilage

- implantable product, implant

- a relatively smooth part of the support medium

- support medium, support bed

- the gross part of the support medium

- chondrocyte cells

rotating the implant to form a spiral cylinder 28 - an arthroscopic delivery device

- arthroscopic delivery device working canal

- arthroscopic delivery device access channel

- injection channel of arthroscopic delivery device 36 - withdrawal and replaceable needle of arthroscopic delivery device

- a telescopic moving handle of the arthroscopic insertion device

- a cap (bell) of rubber or other suitable arthroscopic inserter material

an outwardly deflected holding element of the arthroscopic inserter

- subchodral layer

- well after removal of defective cartilage

- glue

- clamp for fixation of implant ρτ>

- implant fixation clamp head

- intramedullary implant fixation clamp channel 56 - axis, implant fixation clamp carrier 58 - implant fixation clamp ring 60 - separate access channel to implant site 62 - hemostatic barrier in or below the subchondral layer

Claims (1)

PATENT CLAIMS An article comprising a membrane, characterized in that it has at least one layer with a porous surface and comprises submucosal intestinal tissue and cells that adhere to said layer. Second product stated according to claim 1, characterized by the cells are chondrocyte cells. team, that Third product listed according to claim 1, characterized by the membrane does not contain cells. team, that 4th product listed according to claim 1, characterized by the membrane is collagen. team, that 5th product listed according to claim 1, characterized in that the membrane is collagen type I and collagen type III. that 6th product listed according to claim 1, characterized by the membrane is able to resorb again. team, that 7th product stated according to claim 1, characterized by chondrocyte cells are autologous. team, that 8. The product of claim 1, wherein: further comprising a biocompatible adhesive adjacent to said membrane. 9th product according to claim 1, characterized by team, that said membrane being adapted to cover the site of the articular cartilage defect. 10th 2. The article of claim 1 wherein said membrane is adapted to be inserted into a defect in articular cartilage. 11th The product of claim said cartilage membrane. 1 characterized with laid over the defect and being articular The article of claim 1 wherein said membrane is inserted into an articular cartilage defect.