CZ20033051A3 - 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

Method of autologous transplantation

Technical field

The present invention relates to the transplantation of chondrocyte cells, bone and cartilage grafts, healing, joint treatment and 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 US, more than 500,000 arthoplastic operations and total joint replacements are performed annually. Approximately the same number of similar operations is carried out in Europe. This includes about 90,000 total knee joint replacements and about 50,000 knee defect operations (A. Praemer, S. Fumer, D.P. 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 regenerative cartilage treatment and could be performed at an early stage of joint damage, thereby reducing the number of patients in need of artificial joint replacement surgery. With such preventive treatments, the number of patients with developed osteoarthritis would also be reduced.

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 diseased cartilage and subchondral bone are removed and leaving the vascularized spongy bone exposed (J. Insall: Clin. Orhop. 1974, 101, 61; RP Ficat et al., Clin Orthop. 1979, 144, 74; L. Johnson in: Operative Arthroscopy, (JB 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 involvement 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 ultimately 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 patient's knee joints. Kolettas et al. in addition, they investigated the expression of cartilage-specific molecules, such as collagens and proteoglycans, in long-term cell culture. They found that despite morphological changes during cultivation in monolayer 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 the spherical cell morphology), the expressed markers, such as collagen types II and IX and large aggregating proteoglycans, aggrecan, versican, and binding protein, have not changed (E. Kolettas et al., J. Cell Science, 1995, 108, 1991).

Articular chondrocytes are specialized mesenchyme-derived cells found exclusively in cartilage. Cartilage is an avascular tissue whose physical properties depend on the extracellular matrix produced by chondrocytes. During endochondral ossification, chondrocytes undergo maturation leading to cellular hypertrophy, characterized by the onset of expression.

R. Olsen in: Cartilage

Newman) CRC Boca Raton

Dev. Biol. , 1991, 148, type X collagen (W. B. Upholt and R.

Molecular Aspects (editors: B. Halí and 1991, 43; E. Reichenberger et al .:

562; T. Kirsch et al., Differentiation, 1992, 52, 89; M.

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

Despite advances in chondrocyte cultivation and bone and cartilage repair, there has been little success in attempting to transplant cartilage or chondrocytes to repair joint articular surfaces. The teachings of the present invention provide factual and effective means of introducing cartilage and / or chondrocyte transplantation into a joint joint disorder 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 maternal bed or bed that is capable of promoting cell growth and binding thereto, and a method of implanting such an implant to regenerate cells at an implanted site. It is an object of the present invention, in one embodiment, a method of effectively treating the articular cartilage surface of an animal by transplanting an implant comprising chondrocyte cells retained in an absorbable support matrix.

In one embodiment, the chondrocyte cells retain only at the edge of the bed. In one embodiment, the support matrix is a cover bed prepared from collagen and the chondrocyte cells are autologous or homologous. In another embodiment, the support matrix is prepared from collagen and elastin or from collagen and one or more other resorptive materials.

• ·

In another embodiment, the support matrix is prepared from the small intestine submucosa of animal origin.

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

The implant is preferably secured to the transplanted site by means of adhesive or mechanical gripping means. The present invention also provides an instrument for positioning and handling the implant at the implantation site and a clamping device 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 matrix and a method for producing the same.

In another embodiment, the present invention provides a method of treating the articular cartilage surface 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, according to one embodiment, to use certain products that inhibit the formation of vascular tissue, for example, such as capillary capillaries protruding into cartilage that has formed during the process of autologous transplantation of chondrocytes into cartilage defects. Such products are beneficial in repairing cartilage defects in bones where the defects spread to or below the subchondral layer, sometimes referred to as a full thickness defect. The formation of vascular tissue from bone lying beneath 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 may cause interference and excessive overgrowth 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.

Surprisingly, we have found that in a situation where inhibition of the re-vascularization of the cartilage is desired, a hemostatic material can be used which will act as a gel-like artificial coagulate. Giant. 1C depicts a hemostatic barrier 1 that can be used to inhibit re-vascularization of a cartilage defect. If red blood cells were present in the full thickness defect in the articular cartilage, which is covered by such a hemostatic barrier, these red blood cells would chemically turn into hematin and would thus not be able to induce vascular growth.

The haemostatic product used as a re-vascularization inhibition barrier, 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 maternal soil, for example a cell-free maternal soil or other maternal soil described below, is used as a patch overlapping or inserted into the defective area of the joint into which the cultured

chondrocytes are 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. Giant. 2 shows the support maternal soil% which can be used as patches to cover the defective area, and FIG. 3B shows the location of the cartilage defect 18.

Thus, in one embodiment, the present invention provides methods for effectively repairing or treating cartilage defects on the surfaces of the articular bone connection, comprising providing an agent or composition to prevent vascular invasion of the cartilage site to be repaired, and also maternal soil, 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, one that is effective to inhibit vascularization to the site to be repaired; and once the chondrocytes to be placed in the site to be repaired 2 overlap the repaired site so that the chondrocytes are held in place, but still have access to nutrients.

transplant, maternal soil

Particular 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 chondrocyte cell growth, tests maternal beds for use as chondrocyte-retaining implants, and tests each bed's ability to inhibit vascularization as it happens in vivo, where capillary loops protrude into the cartilage formed during the process of autologous transplantation of chondrocytes into cartilage defects.

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 hemostatic material fulfills the objectives of the methods of the present invention by preventing vascular and cellular invasion of developing cartilage to optimize cartilage formation and achieve full thickness repair of any articular cartilage disorders. The hemostatic barrier will preferably be stable for a fairly long period of time sufficient to allow for overall cartilage repair and then to be able to absorb or otherwise break down over time. One material that has found these properties is called Surgicel® W1912 (GG3DH Group, Ethicon Ltd. UK), and is an absorbable hemostat such as oxidized regenerated sterile cellulose.

The present invention also includes an implantable cartilage repair article, method and apparatus for such an implant. The implant comprises supportive matrix 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 maternal bed. The support maternal soil is generally 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. Transplantation can be performed by arthroscopic minimally invasive or open surgery techniques. The method of the invention also encompasses J5 using suitable allogeneic and xenogeneic chondrocyte cells to repair cartilage disorder.

Such an implant is shown in Fig. 4

more specifically, it comprises a support matrix 22 in which chondrocyte cells 24 are stored. A suitable support matrix 22 is a solid or gel-like skeleton that characterizes the ability to maintain a stable shape for as long as the chondrocyte cells are allowed to grow there as before. transplantation, and thereafter, and which provides a natural environment-like system for chondrocyte cells to optimize chondrocyte cell growth differentiation.

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

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

In one embodiment, the support matrix 22 is formed from polypeptides or proteins. The polypeptides or proteins are preferably obtained from natural sources, e.g., 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 matrix 22. Also, it is preferred that the support matrix 22 be reversibly shaped while being handled by the user so that the implant 20 can be manipulated and then returned to its original shape as described below in one aspect of the present invention.

A preferred material from which the support maternal soil is formed is collagen, for example obtained from horses, pigs, cattle, sheep or chickens.

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

Collagen is otherwise obtained from sponge as described in Swatschek et al., Eur. J. of Pharmaceutics and Biopharmaceutics, 2002, 53, 107-113 and in Australian Patent Application No. AU 3741701. Briefly, collagen is isolated from a marine sponge by repeatedly washing the sponge with water, grinding and homogenizing the sponge tissue, treating the solution with high urea concentrations, centrifuging the resulting solution and precipitating collagen from the supernatant.

For example, the implant described above can be prepared by culturing chondrocyte cells in this maternal soil, as described in more detail below.

To prepare an autologous implant, the cartilage biopsy is first collected by arthroscopic techniques 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 binding agent to isolate and extract cartilage chondrocyte cells. The extracted chondrocyte cells are then cultured in a nutrient solution from an initial cell number of about 50,000 to a final number of about 20 million or more chondrocyte cells.

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 matrix 22 and propagate to form an implant 20. Preferably, the chondrocyte cells adhere only to one edge of the outer layer of support matrix 22. implants the patient at the site of the cartilage disorder 18.

It is to be understood that the disorder or injury 18 can be directly treated, slightly enlarged, or eliminated by the surgical procedure prior to implantation, as described in US Patent Application No. 09 / 320,246, to accommodate implant 20. Culture method as well as nutrient and transplant solutions are described in detail below by way of example, beginning with a description of a laboratory procedure for processing the collected cartilage biopsy and culturing chondrocyte cells, in accordance with the present invention.

Culture media (hereinafter "nutrient solutions") used for handling cartilage biopsy during the culture process and for the growth of cartilage chondrocyte cells are prepared by mixing

2.5 mL gentomycin sulfate (70 micromol / liter concentration), 4.0 mL amphotericin (2.2 micromol / liter concentration; Fungizone® trademark, fungicide available from Squibb), 15 mL L-ascorbic acid (300 micromol / liter) liter), 100 mL of fetal calf serum (20% final concentration) and make up with DMEM / F13 to prepare 400 mL of nutrient solution. (The same nutrient solution is also used to transfer cartilage biopsy from a hospital to a laboratory where chondrocytes are extracted and multiplied.)

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 procedure and in transplantation.

The cartilage biopsy previously obtained from the patient for autologous transplantation is sent in the nutrient solution described above to the laboratory for cultivation. In the laboratory, the cartilage biopsy is separated by decantation from the nutrient solution and is 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 preferred is a slightly larger amount, such as about 200 to 300 mg. After weighing, the cartilage is placed in a mixture of 2 mL of collagenase (digestive enzyme; 5000 enzyme units concentration) with approximately 50 mL of pure DMEM / F12 solution, and ground to partially digest the cartilage. After comminution, the comminuted cartilage is transferred through a funnel into a flask and about 50 mL of collagenase and pure DMEM / F12 are added. The milled cartilage is then incubated at 37 ° C and 5% CO 2 for 17-21 hours.

In one embodiment, the minced 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. Thereafter, 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 in a patient, 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 L-ascorbic acid (300 micromoles / liter), 25 mL of autologous blood serum (10% final concentration) and made up with DMEM / F12 to prepare about 300 mL of transplant solution.

The support matrix 22 is then cut 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 (eg, 3-10 million chondrocyte cells) in approximately 5-10 mL of the transplant solution is then soaked in support mother soil 22 and incubated for approximately 72 hours at 37 ° C and 5% CO. ₂ , chondrocyte cells are allowed to grow further. During this incubation, the chondrocyte cells fold into clumps and adhere to the support matrix 22. In one embodiment, the chondrocyte cells adhere to only one outer layer of one side of the support matrix 22. Using this method, the support matrix 22 has been found to promote growth and retention in itself a sufficient number of chondrocyte cells to form the implant 20 without significant loss of biomechanical properties. The supporting maternal soil 22 also promotes continuous growth of the chondrocyte cells upon implantation of the implant 20 at the cartilage defect site 18.

Supporting Maternal Soil 22 provides an environment to

In another embodiment, after a 17 to 21 hour incubation period and determination of cell number and cell viability, as discussed above, the chondrocyte cells are transferred to the transplant solution and then grown directly on support matrix 22 as described above for at least two weeks.

It has been found that the implant 20 can be temporarily deformed without mechanical damage or loss of chondrocyte cells adhering to the support matrix 22. This deformation is completely reversible once the implant 20 has been 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 matrix 22 on which chondrocyte cells grow or have been introduced in sufficient numbers may be temporarily deformed in a manner that allows it to be introduced into the arthroscope working apparatus without mechanically destroyed or without loss of the chondrocyte cells deposited thereon.

At the same time, it has been found that this support maternal soil can be attached to the cartilage defect site by adhesive or mechanical gripping means without interfering with further in-situ differentiation of the chondrocytes and regeneration of the natural cartilage bed material.

Other aspects of the present invention include tools for positioning the implant 20 at the implant site and a mechanical gripping device to maintain the implant 20 at the implant site.

In one embodiment of the present invention, the implantation procedure is performed using arthroscopic techniques. Giant. 5 shows how the implant 20 can be rolled along its length 25 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. 6.

The arthroscopic insertion device 30 in Fig. 6 includes a working channel 32 having a suitable length 35 and a diameter 31 to penetrate the joint and deliver the implant 20 with the desired size. For example, for most processes the diameter is up to 20 mm and the length 35 is about 30 to 60 cm longitudinally relative to the working channel 32. Inside the working duct 32, a movable injection duct 34 with a retractable and replaceable needle 36. The injection duct 34

Attaches to the shaft 38, which is telescopically compressible, at least in part, into the working channel 32. The needle 36 extends the length of the injection channel 34 and allows liquids to pass through it to the the implant site. The injection duct 34 is moved within the working duct 32 by means of a telescopically movable shaft 38 toward or away from the implantation site.

The insertion device 30 also includes a cap, a bell 40 of rubber or other suitable material slidably mounted on the insertion device 30. In use, the cap 40 surrounds the cartilage failure site and does not allow liquids such as blood and other natural liquids to flow into the cartilage failure site. The delivery device 30 also has two or more outwardly deflected gripping members 42: attached to the shaft 38 to grip, insert and position the implant 20 at the implantation site. As the handle 38 moves telescopically toward or away from the user, the gripping members 42 retract within the working channel 32 and move towards each other in a gripping manner (while the shaft 38 moves towards the user) and release the grip ( while the shaft 38 moves away from the user). Such telescopic movement can be controlled by an inclined element (not shown in the figure) located within the shaft 38 which allows the injection channel 34 and the gripping elements 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 failure, preferably deep above the subchondral layer 44, thereby forming a well 46 (see Figs. 8 and 9). ). After removal of the defective cartilage 18, the failure site is ready for insertion of the implant 22. If the subchondral layer is so disturbed that bleeding occurs at the implantation site, the site may optionally be covered with any absorbable material that serves as a hemostatic barrier.

Alternatively, site preparation may include injecting living tissue-compatible adhesive through needle 36 into well 4. 6. Such living tissue-compatible adhesive, designated as adhesive 48 in Figure 8, may comprise an organic fibrin adhesive (e.g., Tissel®, fibrin-based adhesive). , Baxter, Austria, or fibrin glue 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 gripped by the gripping elements 42 and held at the end of the arthroscopic delivery device 30. Arthroscopic delivery device 30 holding the implant 20 at its end is then inserted to the implantation site through the access channel 33, the implant is released from the gripping elements 42 and unfolded by the gripping elements 42 or allowed to unfold as it exits the working channel 32. The access channel 33 includes one or more channels 60 that allow the instruments, such as the insertion device 30 and the observation device, have access to the transplantation site. Using the gripping elements 42, the implant 20 is manipulated such that the chondrocyte-bearing side, in this embodiment, the coarse side 23 of implant 20, faces the well 46 and is gently held in place in the well 46 to allow adhesive 48 to solidify and hold the implant. 20 in well 46.

As shown in Fig. 7, a second access channel 60 having one or more channels can be used to allow access of instruments to the site to assist in the placement of the implant, adhesive and / or gripping mechanical means, or to allow observation devices to be accessed. to the implantation site. Such a separate access channel 60 can also be used for 3 separate channels. ··· "·"··> * _ν used to meet one or more of the functions described in relation to arthroscopic introducer 30 or other arthroscopic instruments.

In another embodiment (Fig. 9), mechanical clamping means such as absorbable clamps, staples, screws or stitches are used to secure the implant 20 in the well 46. Suitable clamps 50 include Ortho-Pin ™ (a commercially available lactide copolymer clamp, Ed. Geistlich Sohne, Switzerland). Giant. 10 illustrates one embodiment of the absorbable clip 50. In this embodiment, the clip 50 comprises a head 52, an intramedullary passage 54 within the axis 56, and one or more retaining rings, 58. The dimensions of the clamp 50 will vary depending on 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 channel 54 has a diameter of about 1.2 mm, the axis 56 has a diameter of about 2 mm, the rings 58 have a diameter of about 2.5 mm. The retaining rings 58 serve to anchor the clamp 50 in a healthy cartilage that surrounds the cartilage disorder. The clamp 50 is made of any material that will not harm the body and is capable of absorbing or otherwise being degraded by the body over a period of time. For example, the clamp 50 may be made of polylactide.

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

As indicated above, in cases where cartilage disorder 18 extends to or below subchondral layer 44, or requires cartilage removal to or below subchondral layer 44, as shown in Figures 11 and 12, the above process may be modified to to include the placement of the hemostatic barrier 62 in the well 46 prior to insertion of the implant. ·· ··· ·· *

In such defects, the physician may optionally apply a hemostatic barrier 62 to inhibit the 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. Appropriate hemostatic barriers will inhibit vascularization and invasion of cells into developing cartilage to optimize cartilage formation and achieve full-thickness cartilage growth at the site of the disorder. The hemostatic barrier is preferably stable for a long 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 matrix 22 or cover bed 2 of the present invention is formed from a collagen membrane as manufactured.

No. 5,028,695 (granted to Entwicklung von by reference.

according to the procedure described in US Patent No. Chemokol Gesellschaft Kollagenprodukten) which is incorporated herein by reference

The collagen membrane can be prepared from crude bovine or porcine 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, reacted with acid, washed with water, left again react with strong alkali, then with acid, causing swelling, react with inorganic salt to shrink, squeeze material to dry weight of 4050% by weight, retained water in this material to remove • »• · · * · · 9 9 material can also be 9 9 material can also be 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 By adding a solvent, if necessary, it can be crosslinked and then dried in a stretched form.

The support matrix 22 or cover bed 2 can 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. Patent No. 5,397,353 (granted to Oliver et al., University of Dundee), is incorporated herein by reference. Collagen / elastin membrane collagen may be obtained from equine, porcine, bovine, ovine and chicken sources. In one embodiment, the support matrix 22 or cover bed 2 is collagen / elastin pork skin that has undergone a number of organic extractions to remove fat content therefrom. Once the fat has been removed, the sections are subjected to a series of enzymatic 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 Permacol ™ -like membranes may be used in the present invention, for example, Rapi-Seal Patch (Fusion Medical Technologies, Inc, Fremont, CA) and Tissue Repair Patch (Glycar Vascular Inc, Dallas, TX).

The collagen / elastin membrane may be up to 20% crosslinked with polyisocyanates, preferably hexamethylene diisocyanate (HMDI). The collagen / elastin membrane used as a support matrix 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 previously described. The implantable article may be placed at or over the cartilage defect site.

In another embodiment, the support matrix 22 can be made from small intestine submucosa (SIS - Smali Intestine Submucose). A method for preparing SIS from the small intestine segment is described in detail in U.S. Patent No. 4,902,508, which is incorporated herein by reference. The intestinal segment, preferably pig, sheep or bovine intestines, is first scraped by longitudinal wiping movements to remove both the outer layers (especially tunica serosa and tunica muscularis) and the inner layer (at least luminal portions of tunica mucosa). The small intestinal mucosa is typically dipped in brine and optionally stored in a hydrated or dehydrated state before being used as described below. A plurality of superimposed layers of small intestinal mucosa tissue are then compressed, pressed against each other, and shaped to provide a multilayer remodeled structure as described in US Patent Nos. 5,788,625, 5,922,028 and 6,176,880 (all granted by DePuy Orthopaedics (Warshaw, IN)), which are incorporated herein by reference. The multilayer structure is provided with a plurality of submucosal layers to form a reconstructive tissue implant structure with the desired thickness to replace the endogenous cartilage structure.

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

Orthopaedics.

Another membrane that can be used for the support matrix 22 or cover bed 2 in accordance with the present invention is the resorbable collagen membrane described in US Patent No. 5,837,278 (granted to Ed. Geistlich Sohne AG), which is incorporated herein by reference. This resorbable 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 alkali-sensitive substances, by reaction with an acid to degrade acid-sensitive substances, by washing, drying, defatting and, if necessary, crosslinking.

Other collagen membranes that can be used as a support matrix 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, ΜΆ). 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 a support matrix 22 or cover bed 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 pig skin (v

essentially pure collagen) and BIOBAR ™ from Col-Bar, Ltd. (RamatHasharon, Israel) are also useful materials for the support mattress 22 or cover bed 2 in the present invention.

Collagen membranes with arranged fibers on macromolecules! These membranes are described, for example, in International Patent Publication No. WO 02/09790 to Mediolanum Pharmaceuticals S.P.A. and Opocrin S.P.A ..

In another embodiment, the support matrix 22 and / or the cover bed 2 may be formed from collagen fibers that crosslink each other through a reducing sugar or a derivative thereof, as disclosed, for example, in US Patent No. 5,955,438 (granted 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, erythrosis, lyxose, xylose, arabinose, allose, altrose, glucose, mannose, gulose, idose, galactose, diatose, triacose, triacose, triacose, triacose , tetrose, pentose, hexose, septose, octose, nanose or docose, and combinations of one or more of these carbohydrates. The support maternal soil 22 and / or cover bed 2 may also include antimicrobials that have a therapeutic effect during cartilage regeneration. Such antimicrobials 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 matrix 22.

• · · · · · · ·

Other collagen materials useful in the present invention are disclosed in US Patent Nos. 5,256,418 and 5,256,418.

No. 5,993,844 (issued to Organogenesis, Inc.), U.S. Pat.

6,206,931 (issued to Cook Biotech, Inc.) and U.S. Pat.

No. 5,026,381 (issued to Colla-Tec Inc), all of which are incorporated herein by reference, and which may belong to one or more of the products discussed herein.

Products not yet marketed in the US that can be used as a support matrix 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 matrix 22 or cover bed 2 may 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 incorporated herein by reference as link. The collagen porous mass may be obtained from commercial sources, for example from Pentapharm AG of Basel, Switzerland; Otto Suwelak Gmg Hof in 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 carried out prior to lyophilization or redissolution of lyophilized collagen in a solution of taurolidine or taurultam and re-lyophilization.

In a further embodiment, the support matrix 22 or cover bed 2 may be formed in accordance with the method for producing the 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 at least partially solidify by cooling the mixture between two embedded surfaces which can be tempered and have different temperatures. An ordered structure is formed during solidification. The partially solidified product is then freeze-dried to give a homogeneous porous structure.

The support matrix 22 or cover bed may also be formed from one or more bioabsorbable polymers, such as collagen, fibrin, laminin, and fibronectin, having large, interconnected pores, in accordance with the method described in US Patent No. 5,869,080 (granted to Johnson &Amp; 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 matrix 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 matrix 22 or cover bed 2 may be made of a bioabsorbable porous mass in accordance with the method described in US Patent No. 5,700,476 (Johnson & Johnson Medical Inc, NJ), which is incorporated herein by reference. The porous mass includes the structure of the bed and at least the bed structure and at least the bed structure. One substructure and at least one pharmacologically active agent. The bed and substructure may, among others, be made of bioabsorbable materials such as 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, but is not limited to, an antimicrobial, a cytokine, a growth factor, or an antibody.

In another embodiment, the support matrix 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 containing 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. Patent No. 5,489,304 (Orgill et al.), Which is incorporated herein by reference in its entirety.

The support matrix 22 or cover bed 22 may also be formed from a multilayer 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. WO 97/00511. 2002/0013627 (Geistlich et al), which are incorporated herein by reference. The bedding layer has an open sponge-like texture and the barrier layer has a closed, relatively impermeable texture. The bedding layer may additionally comprise glycosamine glycans, including, but not limited to, hyaluronic acid, chondroitin sulfate, keratan sulfate, and dermatan sulfate. The bedding layer may be fibronectin calcium alginate or 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, the copolymers also comprising laminin, anchorin II and growth glycolide and lactide, polyorthoesters and polycaprolactones.

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

In a further embodiment of the invention, the support matrix 22 and / or the cover bed 22 is formed of 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 the use of an in vitro system to study the behavior of chondrocyte cells in contact with different carrier beds. These in vitro assays predict the eligibility 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 may be better understood from the following examples, which are intended to illustrate and not limit the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Giant. 1A is a drawing depicting a typical articular bone termination 2- Bone material is typically coated on the surface of the joint with a cartilage cap. If the cartilage cap is damaged or injured (a depression in the cartilage cap 18 in FIG. 1B), the damaged site 18 may be treated directly or somewhat expanded by surgical procedures. Optionally, a haemostatic barrier may be placed in the damaged cartilage cap site to inhibit or prevent vascularization into the recovering cartilage from the underlying bone (Fig. 1C) when necessary, for example, in disorders that extend to the subchondral layer or below her. The chondrocytes to be implanted into the cavity of the disorder are then layered on top of this hemostatic barrier 1 or directly on top of the lesion.

Giant. 2 is a drawing depicting a treatment disorder (gap in the dotted portion) in a cartilage cap (dotted portion) covered with a bed that is used to form a cap / 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 attached differently. The bed covers the damaged area of the joint into which the cultured chorondrocytes were transplanted.

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

Giant. 3B shows cartilage defect or cartilaginous cap injury of the articular end of bone 18.

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

Giant. 5 shows how the implant 20 of FIG. 4 can be used for implantation using an arthroscopic delivery device 30, for example, as shown in FIG. 6.

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

Giant. 7 is a diagram schematically illustrating the placement of the implantable article (implant) 20 of FIG. 5 in place of a defect or injury 18 in the cartilage cap 16 by two access channels 33 and 60 that can accommodate arthroscopic instruments.

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

Giant. 9 is a schematic cross-sectional view of cartilage 16 with damage or injury that does not extend to the subchondral layer 44 and implant 20 that attaches to the failure site by the mechanical clamping device 50.

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

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

Giant. 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 site of failure by the mechanical clamping device 50.

Giant. 13A is a black and white copy of a color photomicrograph of a histological preparation at the onset of growth of chondrocyte cells on a solid support matrix.

• · · · · · · · · · · · · · ·

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

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

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

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

Giant. 13D is a photograph showing collagen support maternal support in which chondrocyte cells grow in a bioreactor system, visualized by immunohistochemical staining.

Giant. 14 is a photomicrograph showing chondrocyte cells on DePuy support matrix.

Giant. 15 is a graph showing the total number of cells in the control (shaded in black), on the Chondro-Gide membrane (dotted), and on the DePuy group (shaded in gray) 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.

Giant. 16 is a graph showing cell viability (percentage) in control (shaded gray), Chondro-Gide membrane (dotted) and DePuy membrane (shaded gray) after three days, two weeks, and six. • t • weeks.

DETAILED DESCRIPTION OF THE INVENTION

EXAMPLE 1

In order for Surgicel® to be used in accordance with our invention in preventing the development of blood vessels into autologically implanted cartilage or chondrocytes, we have allowed Surgicel® to react with a fixative such as glutaraldehyde; we chose to react 0.6% glutaraldehyde with Surgicel® for 1 minute, followed by a wash to remove glutaraldehyde residues that might otherwise be toxic to the tissue. Alternatively, Surgicel® was treated with a fibrin glue called Tisseel® (Immuno AG, Vienna, Austria) prior to reaction with glutaraldehyde as described in Example 2. We have found that Surgicel® fixed, for example, with a fixative such as glutaraldehyde, washed with sterile physiological saline (0.9%) and stored in the refrigerator does not dissolve for one to two months.

Surgicel® generally resorbs 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 time is needed before implanted chondrocytes, embedded in a 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 adequately inhibit vascularization. Therefore, the product should not be absorbed significantly before that time. On the other hand, resorption is ultimately needed.

Therefore, the organic material used as an inhibiting barrier must have these capabilities and we have found that Surgicel® • · • ·

processed 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. 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% glutaraldehyde 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 Surgicel® thus treated 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 to wet 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 is acceptable for use in humans (N. Ihara et al., Burns Inci. Therm. Inj., 1984, 10, 396). The invention also envisages any other adhesive component that may be used in place of the fibrin adhesive. In this • ·

For example, we used Tisseel® or Tissucol® (Immuno AG, Vienna, Austria). The Tisseel® kit consists of the following components:

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

The Tisseel® Suprava contains the DUPLOJECT® application system. The fibrin adhesive or two-component sealant using the Tisseel® kit is mixed as described in the enclosed Immune AG instructions.

EXAMPLE 3

Chondrocytes were cultured in minimal stock culture solution containing HAM F12, 15 mM Hepes buffer and 5-7.5% autologous serum in a CO 2 incubator at 37 ° C and processed in Class 100 laboratory at Verigen Europe A / S, Symbion Science Park, Copenhagen, Denmark. Other culture solution compositions can be used for chondrocyte culture. Cells were trypsinized with trypsin EDTA for 5-10 minutes and counted using trypan blue staining of viable cells in a Burker-Tiirk 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.

The Surgicel® hemostatic barrier was trimmed to a suitable size that fits to the bottom of the well in a NUNCLON ™ tissue culture plate. In this case, a circle with a diameter of approximately 4 cm (but could be of any size) and inserted under aseptic conditions at the bottom of the NUNCLON well ™ tissue culture plates. 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 hemostatic barrier was then washed several times with distilled water and then several more times until the unreacted glutaraldehyde was washed off. A small amount of serum-containing cell culture solution was used to absorb the hemostatic barrier while keeping the hemostatic barrier moist at the bottom of the well.

Approximately 10 ⁶ cells in 1 mL of culture medium were placed directly on top of the hemostatic barrier, dispersed 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 so that the pipette tip was held tangentially to the wall of the well at the outlet of the solution to prevent cells from flushing 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 on the hemostatic barrier began to grow and form clumps. Some cells died due to exposure to low pH before adjusting its value. The plate was incubated for 3 to 7 days with solution exchange on the third day.

At the end of the incubation period, the solution was decanted and the plate was frozen by cooling upon addition of 2.5% glutaraldehyde containing 0.1M dimethylarsinic acid sodium salt, also called sodium cacodylate, with a pH adjusted to pH 7.4 as a fixative (hemostatic barrier) microscopy.

for a cell preparation for later preparation; and

for as electron carrier

EXAMPLE 4

Chondrocytes were cultured in minimal stock culture solution containing HAM F12, 15 mM Hepes buffer and 5-7.5% autologous serum in a CO 2 incubator at 37 ° C and processed in Class 100 laboratory at Verigen Europe A / S, Symbion Science Park, Copenhagen, Denmark. Other culture solution compositions can be used for chondrocyte culture. Cells were trypsinized with trypsin EDTA for 5-10 minutes and counted using trypan blue staining of viable cells in a Burker-Tiirk 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. Tisseel® was applied to both sides of Surgicel® using the DUPLOJECT® system, covering both sides of Surgicel®, the intended patches, 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 tissue culture solution containing serum was used to be absorbed into the hemostatic barrier. Approximately 10 ⁶ cells in 1 mL of tissue culture solution containing

The serum is then placed directly on top of the hemostatic scattered on the surface of the hemostatic barrier. The plate was incubated in a CO 2 incubator at 37 ° C for 60 minutes. An amount of 2 to 5 mL of tissue culture solution containing up 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 to prevent cells from flushing with serum. After 3 to 3 days, microscopic examination showed that the cells adhered to and grown in a satisfactory manner, suggesting that Surgicel® does not exhibit toxicity to chondrocytes and that chondrocytes satisfactorily grow into Surgicel®.

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

EXAMPLE 5

Chondrocytes were cultured in minimal stock culture solution containing HAM F12, 15 mM Hepes buffer and 5-7.5% 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 to 10 minutes and counted using trypan blue staining of viable cells in a Bürker-Turk 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 resorbable bilayer membrane that is used as a patch or bandage covering the area of the 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 gamma-radiation. The bilayer membrane has a porous surface and a dense surface. The membrane is made of type I and type III collagen without further crosslinking or chemical treatment. Collagen is resorbed within 24 weeks. The membrane retains its own structural integrity even when it is wet and can be fixed with stitches or nails. The membrane may also be "bonded with a fibrin glue, such as Tisseel®," to the surrounding cartilage or tissue either instead of the stitch or along with the stitch.

Bio-Gide® was not altered in the Class 100 laboratory and was placed under aseptic conditions on the bottom of NUNCLON ™ tissue culture 6-well Delta Sterile Disposable Cell Research Plates (NUNC (InterMed) Roskilde, Denmark), either with a porous bilayer surface membrane upwards or with a dense membrane surface facing upwards. Approximately 10 ⁶ cells in 1 mL of serum-containing tissue culture medium were placed directly on top of a Bio-Gide ® dispersed on either a 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 to prevent cells from flushing with serum.

On the second day after the chondrocytes were placed in the 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®. However, it was 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 period, the solution was decanted and the plate was frozen by cooling after the addition of 2.5% glutaraldehyde containing 0.1 M dimethylarsinic acid sodium salt, also called sodium cacodylate, with pH adjusted to 7.4 as a fixative for the cell preparation. and as a carrier for Bio-Gide® with cells grown on either a porous or dense surface. 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®, whereas cells cultured 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 stitched 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 area. However, when the dense surface of the collagen patch is directed downwards to 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.

· · 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 ··· ···· ·· ··· ·· ·

EXAMPLE 6

Chondrocytes were cultured in minimal stock culture solution containing HAM F12, 15 mM Hepes buffer and 5-7.5% 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 to 10 minutes and counted using 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 NUNCLON ™ plate was not altered in the Class 100 laboratory.

The Bio-Gide® membrane used as a resorbable 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 Tisseel®, 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 the well of the NUNCLON ™ plate and allowed to solidify completely. A collagen patch such as BioGide® was then applied to the Tisseel® and adhered to the bottom of the well. This combination of Bio-Gide® 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 carrier bed for cartilage formation because the distal surface may be the porous side of the collagen patch to promote chondrocyte and cartilage infiltration. beds. Therefore, this hybrid collagen patch can also be used to mask the collagen. · Top of the implant with a porous collagen surface facing the implanted chondrocytes and as a barrier forming the top. A hybrid collagen patch with an increased aprotinin component can also be used without any organic adhesive, such as Tisseel®, and placed directly in failure with adhesion by natural forces. Thus, the collagen patch can be used both as a hemostatic barrier and as a cell-free covering of the repaired or transplanted site, with the porous surface of the patch oriented to transplanted chondrocytes or cartilage. Another variant would use 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 associations with an organic bed adhesive, wherein the component is similar to a Bio-Gide® bilayer material or glue-like collagen similar to Tisseel®. In another embodiment, in the hybrid collagen patch, no organic adhesive is used to adhere to the repair site.

KIU / mL, in Collagen Resorbable II and Organic

EXAMPLE 7

The kit, as foreseen, will allow suitable practice of 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 appropriate, an appropriate organic adhesive. The kit of the invention may also provide sterile, cell-free bed material as a support for autologous chondrocytes to be implanted in a failure on the surface of the joint. In one embodiment, the kit of the invention comprises a Surgicel® hemostatic barrier and a Bio-Gide® topsheet together with a suitable organic adhesive to coat the Tisseel® hemostatic barrier, wherein Surgicel® and BioGide® have been treated in accordance with the teachings of the invention to extend the time before resorption. 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 is of a semipermeable 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 separate component to be applied as needed due to the inherent variability and uniqueness of the circumstances each repair / transplant operation will encounter.

It will be appreciated by those skilled in the art that numerous modifications 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 a culture solution as described above in a CO ₂ incubator at 37 ° C and processed in a Class 100 laboratory at Verigen Transplatation Service A / S, Copenhagen, Denmark or the University of Ltibeck, Ltibeck, 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 viable staining. cells in tryker blue in the Burker-Tiirk chamber. The cell number was set to 7.5 x 10 ⁵ cells per milliliter. One NUNCLON ™ plate was not altered in the Class 100 laboratory.

Maternal support material, namely Chondro-Gide® Collagen Membrane (identical to Bio-Gide® except that Chondro-Gide® has a greater width and length than Bio-Gide®, both available from Ed Geistlich Sohne, Geistlich Pharma AG, Wolhusen , Switzerland), was cut to a suitable size to fit into the bottom of a well on a NUNCLON ™ cell culture plate. In this case, a circle with a diameter of about 4 cm was placed on the bottom of the well under aseptic conditions.

cells transferred transplant cells in 5 mL

After three weeks, the chondrocyte from the culture solution into the above solution and about 5 x 10 ⁶ chondrocyte transplant solutions were placed directly on top of the support maternal soil 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 arranged in bundles and started to grow on the support maternal soil and could not be removed from the support bed by rinsing it with solution or even mechanically by applying slight pressure to the bed.

At the end of the incubation period, the transplant solution was decanted and the support matrix with chondrocyte cells embedded therein was frozen in 2.5% glutaraldehyde containing 0.1 M sodium dimethylarsinic acid added as fixative. The support maternal 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.

and · · · · · · · «« * * * * * *

EXAMPLE 9

Chondrocytes 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 Transplantation Service A / S, Copenhagen, Denmark, or at the University of Lubeck, Lubeck, Germany. Cells were trypsinized with trypsin EDTA for 5-10 minutes and counted with trypan blue staining of viable cells in a Burker-Turk chamber. The number of chondrocyte cells was adjusted to 5 x 10 ⁵ cells per milliliter. One NUNCLON ”plate was not changed in Class 100 laboratory.

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

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 maternal soil and dispersed on its surface. The plate was incubated for three weeks in a CO ₂ incubator at 37 ° C.

At the end of the incubation period, the transplant solution was decanted and the support matrix with chondrocyte cells embedded therein was frozen in 2.5% glutaraldehyde containing 0.1 M sodium dimethylarsinic acid added as a fixative. The support maternal 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 type II collagen and mouse anti-human aggrecan. Primary antibodies were visualized with fluorescent secondary antibodies. The black and white copy of the color photomicrograph in Fig. 13B shows the chondrocyte cells 24. To better illustrate the features of the photomicrograph, its digitized form is also shown in Fig. 13BB.

During the three-week incubation period on the Chondro-Gide® support matrix, it was observed that chondrocyte cells grew and multiplied on the carrier bed and formed clusters in the center of the carrier bed and lined up 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 a Class 100 laboratory at Verigen Transplantation Service A / S, Copenhagen, Denmark, or at the University of Lubeck, Lubeck, Germany. Cells were trypsinized with trypsin EDTA for 5 to 10 minutes and counted by trypan blue staining of viable cells in a Birker-Tiirk chamber. The number of chondrocyte cells was adjusted to 5 x 10 ⁵ cells per milliliter. One NUNCLON ™ plate was not altered in the Class 100 laboratory.

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

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

maternal soil and dispersed on its surface. The plate was incubated for three weeks in a CO 2 incubator at 37 ° C.

The support matrix with grown chondrocyte cells was then incubated for 16 hours. The support matrix bearing chondrocyte cells was then centrifuged. Cells were plated on a NUNCLON ™ plate and an aliquot was counted by trypan blue staining of viable cells in a Bürker-Türk chamber. Fig. 11C shows a micrograph thereof. The total calculated number of cells 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 test prepared according to US Patent No. 4,902,508 (granted to DePuy, a subsidiary of Ethicon, Inc .; 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 and washed to determine cell number and viability. 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 bottles was 3.5 million cells with 98% viability, respectively. 3.6 million cells with 93% viability.

Cell count 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, two weeks and six weeks. DePuy and ChondroGide® membrane samples were cut to one square inch (i.e., 6.4516 cm ² ) square size. Due to the dry consistency of the DePuy membrane, it was somewhat difficult to cut the membrane.

Chondrocytes were trypsinized to determine cell viability and cell count as indicated above. Cells were centrifuged and resuspended to a concentration of 1 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 well of a culture plate. 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. Additional culture solution (3 mL) was added to each well. The culture plates were incubated for at least three days at 37 ° C.

Giant. 14 illustrates chondrocyte cells 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 to determine cell count and cell count.

viability. Once the membrane had dissolved, the chondrocyte cells were harvested by centrifugation and counted. The duration of action 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 Chondro-Gide® membrane completely dissolved after about one to one and a half hours. Therefore, in order to avoid cell stress, collagenase digestion should not take 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 compared to cells grown on the DePuy and Chondro-Gide ® carrier beds. The viability in the control group, the ChondroGide® cot, and the DePuy cot was high, as shown in Figure 16, 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 on DePuy carrier bed conditions. Therefore, there was no broad 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 consisted of about 72,000 cells, whereas there were about 109,167 cells on the Chondro-Gide ® cot and about 169,583 cells on the DePuy cot. After two weeks, the control cells multiplied about eight times to 575,167 cells, whereas the Chondro-Gide® cells nearly four times to 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 somewhat to 528.333, while the number of cells on Chondro-Gide® and DePuy decreased dramatically to 153.333, respectively. 100.833.

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

Claims (8)

The patent claims has at least one layer with a porous surface and comprises submucosal intestinal tissue and cells that adhere to said layer. 2. The article of claim 1 wherein said cells are chondrocyte cells. 3. The article of claim 1 wherein said membrane is free of cells. 4. The article of claim 1 wherein said membrane is collagen. 5. The article of claim 1 wherein said membrane is collagen type I and collagen type III. 6. The article of claim 1 wherein said membrane is resorbable. 7. The article of claim 1 wherein said chondrocyte cells are autologous. 8. The article of claim 1, further comprising a biocompatible adhesive adjacent said membrane. The article of claim 1 wherein said membrane is adapted to be positioned over a defect of articular cartilage. • · · · · · 10. 11. 2. The article of claim 1, wherein said membrane is adapted to be placed in an articular cartilage defect. The article of claim 1 wherein said membrane is placed over a defect of articular cartilage. The article of claim 1 wherein said membrane is placed in an articular cartilage defect.