Method for providing a kit for autologous transplantation

ES 2 218 697 T3

DESCRIPTION

Kit for the transplantation of chondrocytes in a joint.

Field of the invention

The immediate invention relates to the field of chondrocyte transplantation, bone and cartilage grafting, healing, joint repair and the prevention of arthritic conditions. In particular, methods for preparation of the graft site, instruments for such preparation and for autogenous transplantation of cells to the prepared graft site are described.

Fundamentals of the invention

More than 500,000 arthroplastic procedures and total arthroplasties are performed each year in the United States. About the same number of similar procedures are carried out in Europe. Included in these figures are approximately 90,000 total knee replacements and around 50,000 procedures to repair imperfections in the knee per year in Europe. The number of procedures is essentially the same in the US. (In: Praemer A., ​​Furner S., Rice, DP, Musculoskeletal conditions in the United States, American Academy of Orthopedic Surgeons, Park Ridge, III, 1992, 125). Most useful would be a method for cartilage regeneration treatment and could be carried out at an earlier stage of joint damage, thereby reducing the number of patients requiring joint arthroplasty surgery. With such preventive treatment methods, the number of patients developing osteoarthritis would also decrease.

With the techniques used to regenerate the surface of the cartilage structure in joints, it has been mainly attempted to produce cartilage repair using subchondral perforation, abrasion and other methods by which there is excision of diseased cartilage and subchondral bone, exposing vascularized cancellous bone. (Insall, J., Clin. Orthop. 1974, 101.61; Ficat RP et al., Clin. Orthop, 1979, 144, 74; Johnson LL, In: Operative Arthroscopy, McGinty JB, Ed., Raven Press, New York, 1991, 341).

Coon and Cahn (Science 1966, 153, 1116) described a technique for growing cells that synthesize cartilage from chicken embryo somites. Later, Cahn and Lasher (PNAS, USA 1967, 58, 1131) used the system for analysis of the involvement of DNA synthesis as a prerequisite for cartilage differentiation. Chondrocytes respond to both epidermal growth factor, EFG, and fibroblast growth factor, FGF (both for its acronym in English), by growth (Gospodarowicz and Mescher, J. Cell Physiology 1977, 93, 117), but lastly they lose their differentiated function (Benya et al., Cell 1978; 15, 1313). Methods for chondrocyte growth have been described and are being used mainly with minimal adjustments by Brittberg, M. et al., (New Engl. J. Med. 1994, 331, 889). Cells that were grown using these methods were used as autogenous transplants into knee joints of patients. Additionally, Kolettas et al. (J. Cell Science 1995, 108, 1991) examined the expression of specific cartilage molecules such as collagens and proteoglycans in prolonged cell culture. They found that despite morphological changes during cultivation in single-layer cultures (Aulthouse, A. et al., In Vitro Cell Dev. Biol., 1989, 25, 659; Archer, C. et al., J. Cell Sci. 1990, 97, 361; Hanselmann, H. et al., J. Cell Sci. 1994, 107, 17; Bonaventure, J., et al., Exp. Cell Res. 1994, 212, 97) in comparison with suspension cultures that have grown on agarose gels, alginate beads or as spinner-type cultures (which retain a circular cell morphology) tested by various scientists, the chondrocyte indicator markers did not change, such as type II and IX collagens and the highly agglutinating proteoglycans, aggrecan, versican, and binding protein were unchanged (Kolettas, E. et al., J. Cell Science, 1995, 108, 1991).

Articular chondrocytes are specialized mesenchymal 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 a maturation that leads to cellular hypertrophy, characterized by the onset of type X collagen expression (Upholt, WB and Olsen, RR, In: Cartilage Molecular Aspects (Authors Hall, B and Newman, S) CRC Boca Raton 1991, 43; Reichenberger, E. et al., Dev. Biol. 1991, 148, 562; Kirsch, T. et al., Differentiation, 1992, 52, 89; Stephens, M. et al., J. Cell Sci. 1993, 103, 1111).

Excessive degradation of type II collagen also occurs in the outer layers or articular surfaces of the joints due to osteoarthritis. The collagen network is weakened accordingly and fibrillation subsequently develops whereby matrix substances such as proteoglycans are lost and finally completely displaced. Such cartilage fibrillation with osteoarthritis, weakened, can get down to calcified cartilage and subchondral bone (Kempson, GE et al., Biochim. Biophys, Acta 1976, 428, 741; Roth, V and Mow, VC, J. Bone Joint Surgery, 1980, 62A, 1102; Woo, SL-Y et al., In Handbook of Bioengineering (authors R. Skalak and S. Chien), McGraw-Hill, New York, 1987, pp. 4.1-4.44).

Descriptions of the basic development, histology, and microscopic anatomy of bone, cartilage, and other such connective tissues can be found, for example, in Wheater, Burequipot, and Daniels, Functional Histology, 2^to Edition (Churchill Livingstone, London, 1987, Ch. 4). Descriptions of the basic histology of imperfections in bone, cartilage and other connective tissue can be found, for example, in Wheater, Burkitt, Stevens and Lowe, Basic Histopathology, (Churchill Livingstone, London, 1985, Ch. 21).

Despite advances in chondrocyte culture and bone and cartilage manipulation, there have been little success with attempts to transplant cartilage or chondrocytes for the repair of damaged joint surfaces. The instant invention disclosures provide effective and efficient means of promoting the transplantation of cartilage and / or chondrocytes into a blemish in a joint prosthesis or other cartilage-covered bone surface, whereby cartilage is regenerated to repair the blemish. The immediate description also provides surgical instruments that are designed to prepare the graft site in a manner that facilitates efficient integration of graft material to the graft site.

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Brief summary of the invention

The immediate description provides a new method, which by itself is not part of the invention, for the efficient treatment of cartilage from surfaces of joint prostheses by transplantation of chondrocytes into a suitable matrix, to a surface to be treated, with a hemostatic barrier and a cell-free overlay plate comprising: first, put a hemostatic barrier proximal to the surface to be treated, put chondrocytes in a suitable matrix on the surface to be treated distal to the hemostatic barrier, cover the surface to be treated with a cover plate without cells. A hemostatic barrier, as will be further described below, is a barrier that inhibits or prevents the penetration of vascularizing cells and tissue into the grafted material. In particular, the immediate method provides a hemostatic barrier that is a resorbable, semi-permeable material that inhibits or prohibits vascular infiltration through the barrier. In one embodiment, the hemostatic barrier contains collagen. Cell-free is used herein as in the art and means a material that is substantially free of healthy cells that is capable of further cell division, diffusion, or biological activity. In a preferred embodiment, a cell-free material is free of all healthy nucleated cells. In one embodiment, the immediate method includes the use of a cell-free cover plate containing a semipermeable collagen matrix. In a preferred embodiment of the method, the porous surface of the cell-free cover plate is directed towards the implant material.

The new method further provides for autogenous transplantation of collagen or chondrocytes to a graft site, where the graft site has first been prepared by surgical manipulation to better accept the graft material. In one embodiment, the graft site is sculpted so that the graft site walls are contoured in a wavy pattern so that the graft material, when placed within the graft site, expands to contact the wall. graft site, it will have resistance against removal or expulsion of the entire graft from the graft site. The method may require surgical instruments designed to sculpt the graft site.

The invention provides a kit for transplantation of cartilage and / or chondrocytes on the surface of a joint prosthesis, wherein said kit comprises cells for placement attached with a hemostatic barrier, semipermeable cell-free cover plate and organic glue. In a further embodiment, the kit may optionally further provide one or more surgical instruments that can be used to sculpt the graft site in accordance with the new disclosed methods.

Brief description of the drawings

The present invention will be better understood by examining the following figures illustrating certain properties, in which:

Fig. 1A is a drawing showing a typical hinge end of a bone. Typically, the bone material is covered on the joint surface with a cartilaginous material.

Figure 1B shows an example of where a cartilaginous cap (hole in cartilage) blemish or injury occurs and such blemish can be directly treated, slightly lengthened, or sculpted to accept the grafted material by surgical procedures prior to treatment.

Figure 1C shows how the hemostatic barrier (numbered 1) is placed within the imperfection in the cartilage cap to inhibit or prevent vascularization in the regenerating cartilage, from the underlying bone. The chondrocytes to be implanted in the cavity of the blemish are then stratified on the hemostatic barrier.

Figure 2 is a drawing showing the treated blemish (hole in cartilage) in the cartilaginous cap covered by a cell-free semipermeable material (numbered 2) that is used to form a cap / plate or bandage over the blemish site. This cap is fixed in place, either sutured to the edge of the socket in healthy cartilage or otherwise attached. This cap is covering the imperfect area of ​​the joint where the cultured chondrocyte / cartilage transplant has been placed or will be placed under the partially attached cap.

Figure 3A is a diagram illustrating the differential response to compression and shear forces by harder and softer cartilage with posterior zone of demarcation.

Figure 3B illustrates the graft site, after the blemish has been sculpted to have wavy walls.

Figure 3C illustrates the sculpted graft site with hemostatic barrier (1), transplanted material (3), and cell-free cover plate (2) in place within articular surface cartilage (4).

Figure 4A illustrates an embodiment of the surgical device as part of the kit of the immediate invention, showing cutting teeth (5) and protruding positioning nail (6). The cross-section illustrations to the right show two possible configurations of the cutting blades.

Figure 4B illustrates a second embodiment of the surgical device.

Figure 5 is a diagram illustrating the modified differential response to compression and shear forces by harder cartilage and softer cartilage after sculpting the graft site.

Figure 6A is a Nuclear Magnetic Resonance Imaging of a pig knee showing cartilage imperfection in the left (medial) epiphysis.

Figure 6B is a Nuclear Magnetic Resonance of the same pig knee three months after treatment.

Detailed description of the invention

This invention relates to the use of certain products that inhibit the formation of vascular tissue, such as for example protruding capillary loops in the cartilage that is being established, during the procedure of transplantation of chondrocytes in imperfections in the cartilage. The formation of vascular tissue from the underlying bone will tend to bulge into the new cartilage to be formed leading to a cell appearance distinct from the desired specialized mesenchymal chondrocytes.

Contaminating cells introduced by vascularization can lead to invasion and proliferation in the cartilage that has to be formed by the implanted chondrocytes. One of the types of commercial products that can be used in this invention is Surgicel® (Ethicon Ltd., UK) which can be absorbed

ES 2 218 697 T3 ber after a period of 7-14 days. The use of this material in the new method described is contrary to the normal use of a hemostatic device, such as Surgicel.^®, as described in the Ethicon Ltd.

Surprisingly, we have found that in a situation where it is desired to inhibit revascularization in cartilage, a hemostatic material will act as an artificial gel-like coagulant. If red blood cells should be present within the full-thickness, articular cartilage imperfection, which is covered by such a hemostatic barrier, these blood cells will be chemically changed to hematin, thereby rendering them unable to induce vascular growth. Thus, a hemostatic product used as a revascularization inhibitory barrier with or without fibrin adhesives, such as, for example, Surgicel^®, is efficient for the intended method, as explained by the immediate invention. Another part of this invention is a cell-free component, which is used as a plate covering the imperfect area of ​​the joint where the cultured chondrocytes / cartilage are being transplanted, using autogenous chondrocytes for transplantation. The method also considers the use of allogeneic chondrocytes or xenogeneic chondrocytes suitable for the repair of a cartilage imperfection.

Thus, the immediate description explains methods for the efficient repair or treatment of cartilage imperfections on bone surfaces of joint prostheses, comprising: administering an agent or device to block vascular invasion at the cartilage site to be repaired, and also provide a cell-free barrier that will isolate the repair site and keep the transplanted cells in place. Thus, the instant invention provides a kit comprising a hemostatic barrier component for insertion at the site to be repaired, such that there is efficient inhibition of vascularization at the site to be repaired; and once the chondrocytes to be transplanted are put into the site to be repaired, a cell-free semi-permeable barrier is capped over the repair site so that the transplanted chondrocytes remain in place, but still are able to gain access to nutrients.

Certain aspects of the choice of the components of the kit of the invention have been exemplified using an in vitro system to study the behavior of chondrocytes when they are put in contact with a certain product or a combination of certain products that inhibits the formation of vascular tissue. . This in vitro test predicts the ability of certain tested materials to inhibit vascularization, as will occur in vivo in the event that capillary loops protrude in the cartilage that is being established during the autogenous chondrocyte transplantation procedure in cartilage imperfections.

Suitable hemostatic products will be characterized as having the ability to inhibit the growth or invasion of vascular tissue, osteocytes, fibroblasts, etc., into developing cartilage. A suitable hemostatic material will achieve the objective of the new method in that it should avoid vascular and cellular invasion in the developing cartilage, to optimize cartilage formation and to achieve full thickness repair of any imperfections in the cartilage or articular. Ideally, the hemostatic barrier will be stable for a long enough period of time to allow complete cartilage repair, and will then be capable of being resorbed or otherwise broken down over time. A material identified as suitable is called Surgicel^®W1.912 (a sterile regenerated oxidized cellulose containing absorbable hemostatic; Lot. GG3DH, Ethicon Ltd, UK). Another example of a suitable material is BioGide^® (a commercially available type I collagen matrix plate; Geistlich Sohne, Switzerland).

Suitable organic glue material can be found commercially, such as for example Tisseel® or Tissucol® (fibrin-based adhesive; Immuno AG, Austria), Protein Adhesive (Cat. A-2707, Sigma Chemical, USA), and Dow Corning Medical Adhesive B (Cat. 895-3, Dow Corning, USA).

Surgical instruments considered by the immediate invention can be made of metal and / or plastic suitable for making non-reusable single use or reusable multi-use surgical instruments. The cutting instrument can contain cutting teeth that are completely round or flat, or something in between. As cartilage is a relatively soft material it may be advantageous to fabricate hardened plastic cutting edges that will be capable of sculpting cartilage without being capable of damaging bone. Such cutting instruments can be manufactured to incorporate openings for fluid delivery, suction removal of cutting particles and fluid, and fiber optic strands for illumination and visualization of the blemish site.

Certain aspects of the immediate invention can be better understood as illustrated by the following examples, which by themselves are not all part of the content of the claims and which are meant by way of illustration and not limitation. Example 1

For the Surgicel® to be used in a kit according to the invention, to avoid the development of blood vessels in autogenous implanted cartilage or chondrocytes, the Surgicel was first treated^® with a fixative such as glutaraldehyde. Briefly, Surgicel® was treated with 0.6% glutaraldehyde for 1 minute, followed by various washes to remove glutaraldehyde residues that could otherwise be toxic to tissue. Alternatively, the Surgicel was treated^® with the fibrin adhesive called Tisseel^®, prior to treatment with glutaraldehyde, as described in Example 2. It was found that Surgicel^® Fixed, for example, with a fixative such as glutaraldehyde, washed with sterile physiological saline (0.9%) and stored in the refrigerator, did not dissolve for 1 to 2 months. Surgicel is generally reabsorbed within 7 to 14 days. This time would be too short, because a longer time is needed to avoid the development of blood vessels or vascularization such as that of the bone structure in the implanted cartilage before the implanted chondrocytes have grown in a layer of solid cartilage, achieving their requirements. of nutrition from nearby cartilage. In other words, sufficient inhibition of vascularization is needed for a longer time such as, for example, a month. Therefore, the product should not be significantly absorbed prior to that time. On the other hand, you finally need reabsor

ES 2 218 697 T3 tion. Therefore, organic material used as a barrier inhibition will have these capabilities, and Surgicel has been found to^® treated in this way provides that function.

Example 2

The Surgicel^® it was also coated with an organic glue, in this example the glue used was Tisseel^®, but others can also be used. This product, together with Surgicel^® produces a barrier that can be used for the particular purpose of the kit of the invention. Any other vascular or hemostatic inhibitory barrier could be used. The Tisseel^® mixed as described below. The Surgicel^® was later coated with Tisseel^® by spraying the Surgicel material^® on both sides until soaked. It was allowed after the Tisseel^® (fibrin glue) will solidify at room temperature. Immediately before complete solidification, the Surgicel was then placed^®, coated in 0.6% glutaraldehyde, for 1 minute and then washed with sterile physiological saline (0.9%). The pH was then adjusted by phosphate saline buffer, PBS, and / or with NaOH until the pH was stable at 7.2 to 7.4. Later, the Surgicel® treated in this way was then washed in tissue culture medium, such as minimal essential medium / F12 with 15 mM Hepes buffer.

As mentioned in this example, we have used Tisseel^® as a fibrin adhesive to coat Surgicel^®. In addition, the adhesive or fibrin glue can also be applied directly to the bottom of the lesion towards the bone, where the Surgicel sticks^®. The in vitro system used, rather than in vivo assay, consisted of a 6-well, sterile, non-reusable plate, Delta, from nUnCLON ™, for cell research work (NUNC, InterMed, Roskilde, Denmark). Each well measured approximately 4 cm in diameter.

In the invention, the fibrin adhesive can be any adhesive that together with the fibrin component produces a glue that can be tolerated in humans (Ihara, N et al., Burns Incl. Therm. Inj. 1984, 10, 396) . The invention also features any other glue component that can be used in place of the fibrin adhesive. In a preferred embodiment, we use Tisseel^® or Tissucol^® (Immuno AG, Vienna, Austria). Tisseel's kit^® consists of the following components:

Tisseel^®, a coagulable protein containing lyophilized, virus-inactivated sealant thereof: Fibrinogen, Plasma Fibronectin (CIG) and Factor XIII and Plasminogen.

Aprotinin solution (bovine)

Thrombin 4 (bovine)

Thrombin 500 (bovine)

Calcium Chloride Solution

Tisseel's kit^® contains a DUPLOJECT Application System^®. The fibrin adhesive or the two-component sealant, which is used in the Tisseel kit^®, is combined as follows according to the Immuno AG product insert sheet:

Example 3

Chondrocytes were grown in minimal essential culture medium containing HAM F12 and 15 mM Hepes buffer and 5 to 7.5% autoserum, in a CO incubator.₂ at 37 ° C and handled in a Class 100 laboratory at Verigen Europe A / S, Symbion Science Park, Copenhagen, Denmark. Other culture medium compositions can be used to culture chondrocytes. Cells were trypsinized using trypsin and EDTA, for 5 to 10 minutes, and counted using Trypan Blue viability staining in a Burker-Türk chamber. The blood count was adjusted to 7.5 x 10⁵ cells per ml. A NUNCLON ™ plate was uncovered in the Class 100 laboratory.

Surgicel hemostatic barrier was cut^® to a suitable size, adjusted to the bottom of the well in the NUNCLON ™ tissue culture tray. In this case a circle, approximately 4 cm in size (but could be any size possible) and placed in aseptic conditions at the bottom, in the well, in a sterile, 6-well, non-reusable plate, Delta , from NUNCLON ™, for cell research work (NUNC, InterMed, Roskilde, Denmark). The downhole hemostatic barrier was pretreated as described in Example 1. This treatment significantly delayed Surgicel uptake. This hemostatic barrier was then washed several times in distilled water and several times thereafter, until the unreacted glutaraldehyde was washed away. A small amount of the serum-containing cell culture medium was applied so that it was absorbed into the hemostatic barrier and at the same time kept the hemostatic barrier moist at the bottom of the well.

Approximately 10 were directly put⁶cells in 1 ml of culture medium, on top of the hemostatic barrier, dispersed on the surface of the hemostatic barrier, pre-treated with 0.4% glutaraldehyde, as described above. The plate was then incubated in a CO2 incubator at 37 ° C, for 60 minutes. An amount of 2 to 5 ml of tissue culture medium containing 5 to 7.5% serum was carefully added to the well containing the cells avoiding splashing the cells by supporting the tangential pipet tip next to the well when he was expelling the medium. The pH of the medium was shown to be too low (pH ~ 6.8). The pH was then adjusted to 7.4 to 7.5. The next day, some chondrocytes had started to grow on the hemostatic barrier, arranged in groups. Some of the cells had died due to low pH exposure prior to pH adjustment. The plate was incubated for 3 to 7 days with a change of medium on day 3.

At the end of the incubation period, the medium was decanted and cryo-frozen 2.5% glutaraldehyde containing 0.1 M sodium salt of dimethylarsinic acid was added (also called sodium cacodylate, the pH was adjusted with HCl to 7.4) , as a fixative for cell preparation and support (hemostatic barrier) for subsequent preparation for electron microscopy.

Example 4

Chondrocytes were grown in minimal essential culture medium containing HAM F12 and 15 mM Hepes buffer and 5 to 7.5% autoserum, in a CO2 incubator at 37 ° C and manipulated in a Class 100 laboratory at Verigen Europe. A / S, Symbion Science Park, Copenhagen, Denmark. Other culture medium compositions can be used.

ES 2 218 697 T3 to culture chondrocytes. Cells were trypsinized using trypsin and EDTA, for 5 to 10 minutes, and counted using Trypan Blue viability staining in a Bürker-Türk chamber. The blood count was adjusted to 7.5 x 10⁵ cells per ml. A NUNCLON ™ plate was uncovered in the Class 100 laboratory.

Surgicel was treated^® (for use as a hemostatic barrier) with 0.6% glutaraldehyde, for one minute, as described in Example 1, and washed with sterile 0.9% sodium chloride solution or, preferably, with a buffer such as PBS buffer or the culture medium such as MEM / F12, because the pH after glutaraldehyde treatment is 6.8 and should preferably be 7.0 to 7.5. The Tisseel was applied^® on both sides of the Surgicel^® using the DUPLOJECT system^®, thereby coating both sides of the Surgicel^®, the plate to be used, with fibrin adhesive. The glue was allowed to dry under aseptic conditions, for at least 3 to 5 minutes. The "coated" hemostatic barrier was bottomed out in a NUNCLON ™ Delta 6-well sterile non-reusable plate for cell research work. A small amount of tissue culture medium containing serum was applied to absorb into the hemostatic barrier. Approximately 10 were directly put⁶ cells in 1 ml of tissue culture medium containing serum, on top of the hemostatic, dispersed on the surface of the hemostatic barrier. The plate was then incubated in a CO incubator.₂ at 37 ° C, for 60 minutes. A 2 to 5 ml amount of tissue culture medium containing 5 to 7.5% serum was carefully added to the well containing the cells, avoiding splashing the cells by supporting the tangential pipette tip next to the well. when the medium was being ejected. After 3 to 6 days, microscopic examination showed that the cells were adhering to, and growing in the Surgicel® in a satisfactory manner, suggesting that the Surgicel® did not show toxicity to chondrocytes and that the chondrocytes were growing in a satisfactory in the Surgicel^®.

The plate was incubated for 3 to 7 days with a change of medium on day 3. At the end of the incubation period the medium was decanted and cryo-frozen 2.5% glutaraldehyde containing 0.1M sodium salt of dimethylarsinic acid was added, Also called sodium cacodylate, the pH was adjusted with HCl to 7.4, as a fixative for the preparation of the cell and the support (hemostatic barrier) for subsequent preparation for electron microscopy.

Example 5

Chondrocytes were grown in minimal essential culture medium containing HAM F12 and 15 mM Hepes buffer and 5 to 7.5% autoserum, in a CO2 incubator at 37 ° C and manipulated in a Class 100 laboratory at Verigen Europe. A / S, Symbion Science Park, Copenhagen, Denmark. Cells were trypsinized using trypsin and EDTA, for 5 to 10 minutes, and counted using Trypan Blue viability staining in a Burker-Türk chamber. The blood count was adjusted to 7.5 x 10⁵ to 2 x 10⁶ cells per ml. A NUNCLON ™ plate was uncovered in the Class 100 laboratory.

It has been found that Bio-Gide® can be used as a resorbable bilayer membrane, which will be used as the plaque or bandage that covers the imperfect area of ​​the joint where the cultured chondrocytes are being transplanted as well as the hemostatic barrier. The Bio-Gide^® It is a pure collagen membrane obtained by standardized, controlled manufacturing procedures (by ED: Geistlich Sohne AG, CH-6.110 Wolhusen). Collagen is extracted from veterinary certified pigs and carefully purified to avoid antigenic reactions, and sterilized in double ampoules by γ-irradiation. The two-layer membrane has a porous surface and a dense surface. The membrane is prepared from type I and type III collagen without additional crosslinking or chemical treatment. Collagen is reabsorbed in 24 weeks. The membrane retains its structural integrity even when wet and can be secured by sutures or nails. The membrane can also be "glued on" using fibrin adhesive such as Tisseel.^® to nearby cartilage or tissue either in place of sutures or in conjunction with sutures.

The Bio-Gide^® was uncapped in a class 100 laboratory and placed under aseptic conditions at the bottom of the wells in a sterile 6-well, Delta, non-reusable plate from NUNCLON ™, for cell research work, well with the porous surface of the two-layer membrane facing up or with the dense surface facing up. Approximately 10 were directly put⁶ cells in 1 ml of tissue culture medium containing serum, on top of the Bio-Gide^®, dispersed either on the porous or dense surface of the Bio-Gide^®. The plate was then incubated in a CO2 incubator at 37 ° C, for 60 minutes. A 2 to 5 ml amount of tissue culture medium containing 5 to 7.5% serum was carefully added to the well containing the cells, avoiding splashing the cells by supporting the tangential pipette tip next to the well when the medium was being ejected.

On day 2, after the chondrocytes were placed in the well containing the Bio-Gide^®, cells were examined under a Nikon inverted microscope. Some chondrocytes were observed to have adhered to the edge of the Bio-Gide. It was not possible of course to be able to look through the Bio-Gide^® myself using this microscope.

The plate was incubated for 3 to 7 days with a change of medium on day 3. At the end of the incubation period, the medium was decanted and cryo-frozen 2.5% glutaraldehyde, containing 0.1 M sodium salt of dimethylarsinic acid, was added. , also called sodium cacodylate, the pH was adjusted with HCl to 7.4, as a fixative for the preparation of the cell and the Bio-Gide support^® with well-cultured cells on the porous surface or the dense surface. Bio-Gide plates^® they were then sent for electron microscopy to the Department of Pathology, Herlev Hospital, Denmark.

Electron microscopy showed that chondrocytes grown on the dense surface of the BioGide^® did not grow into the BioGide's collagen framework^®While the cells grown on the porous surface did indeed grow in the collagen structure and also showed the presence of proteoglycans and did not show signs of fibroblast structures. This result showed that when the collagen plate, such as a Bio-Gide plate^®, is sewn as a plate covering a cartilage imperfection, the porous surface will be upside down

ES 2 218 697 T3 jo towards the imperfection in which the cultured chondrocytes have to be injected. They will then be able to penetrate the collagen and produce a smooth cartilage surface in accordance with the healthy surface, and a smooth layer of proteoglycans will accumulate in this area. Whereas, if the dense surface of the collagen is upside down in the blemish, the chondrocytes to be implanted will not integrate with the collagen, and the cells will not produce the same smooth surface as described above.

Example 6

Chondrocytes were grown in minimal essential culture medium containing HAM F12 and 15mM Hepes buffer and 5 to 7.5% autoserum, in a CO incubator.₂ at 37 ° C and handled in a Class 100 laboratory at Verigen Europe A / S, Symbion Science Park, Copenhagen, Denmark. Cells were trypsinized using trypsin and EDTA for 5 to 10 minutes and counted using Trypan Blue viability staining in a Bürker-Türk chamber. The blood count was adjusted to 7.5 x 10⁵ to 2 x 10⁶ cells per ml. A NUNCLON ™ plate was uncovered in the Class 100 laboratory.

Bio-Gide® used as a resorbable bilayer membrane can also be used, along with an organic glue such as Tisseel.^® with significantly higher additional Aprotinin content than normally found in Tisseel^®, as described in the product insert. Increasing the Aprotinin content to approximately 25,000 KIU / ml will delay the resorption of the material for weeks instead of the normal interval of days.

To test this characteristic in vitro, the Tisseel was applied^® to the bottom of the NUNCLON ™ plate and allowed to incompletely solidify. A collagen plate was then applied, such as a Bio-Gide^®, about the Tisseel^® and stuck to the bottom of the well. This combination of Bio-Gide^® and Tisseel® was designed to be a hemostatic barrier that would inhibit or prevent the development or infiltration of blood vessels in the chondrocyte transplant area. This hybrid collagen plate can now be used both as a hemostatic barrier at the bottom of the lesion (the most proximal to the surface to be repaired) as well as as a support for cartilage formation because the distal surface can be the side porous of the collagen plate, and thereby stimulate the infiltration of chondrocytes and cartilage matrix. Thus, this hybrid collagen plate can also be used to cover the top of the implant with the porous collagen surface facing downward toward the implanted chondrocytes and the barrier that is forming the top. The hybrid collagen plate with high Aprotinin component, without any organic glue, such as Tisseel^®, It can also be worn and put inside the blemish directly, adhering by natural forces. Thus the collagen plate can be used both as the hemostatic barrier and as the cell-free coating of the repair / transplant site, with the porous surfaces of the plates facing the transplanted chondrocytes / cartilage. Another variant would use a collagen plate consisting of type II collagen (ie, from Geistlich Sohne AG, CH-6,110 Wolhusen).

Thus, the immediate invention provides a hybrid collagen plate that is separated into two parts in the kit in the event that said plate is a collagen matrix with high levels of aprotinin component, preferably approximately 25,000 KIU / ml, in conjunction with an organic matrix glue, in case the collagen component is similar to Bio-Gide's resorbable bilayer material^® or Type II collagen and the organic glue is similar to the Tisseel® material. No organic glue may be used to adhere the hybrid collagen plaque to the repair site. Example 7

Due to the weakened structure of the arthritic cartilage, adhesion of cultured autogenous chondrocytes, transplanted to an imperfect cartilage graft site, can be inhibited, thereby creating a marginal zone (demarcation zone) between the newly implanted cartilage / chondrocytes. (s) and surrounding established cartilage. This marginal zone will be most pronounced if the graft site is prepared for grafting by creating smooth, straight walls cut in a linear fashion. Shear and compression forces through such a marginal zone (as illustrated in Figure 3A) will exert great force to pull out the graft when the graft site is cut in a linear fashion. This marginal zone and differential displacement of materials along this zone will inhibit confluent healing between the grafted material and the surrounding material. This marginal zone shear is exacerbated when the hardness of the confined material is different. In many cases the graft material is softer than the surrounding material, however, in some cases of osteoarthritis, the surrounding cartilage may, in fact, be softer than the implanted chondrocytes / cartilage.

Therefore, to solve this problem, the new method on which the kit of the invention is based, explains the use of surgical instruments to sculpt the walls of the graft site so that the walls are not linear and thereby provide surfaces corrugated that will reduce the shear of the marginal zone, and provide anchorage for the grafted material. It is also possible to shape the graft site so that the diameter of the site proximal to the bone surface is of a larger dimension than the opening distal to the bone and the cartilage surface to be repaired, so that there is a " inverted funnel ”. A narrow opening in the surface will help reduce marginal zone shear and the expulsion of graft material from the graft site. A preferred embodiment describes sculpting the walls of the graft site in a manner similar to a threaded opening to receive a bolt or screw (as illustrated in Figure 3B), thereby providing mechanical resistance to compression and / or ejection of the grafted material. from the graft site that can be described as "male" and "female" threading.

The surgical instruments considered by the kit of the immediate invention can be made of metal and / or plastic suitable to make non-reusable, single-use, or reusable multi-use surgical instruments. As cartilage is a relatively soft material, it may be advantageous to fabricate hardened plastic cutting edges that will be capable of sculpting cartilage without being capable of damaging bone. Such cutting instruments can be manufactured to incorporate openings for fluid delivery, suction removal of cutting particles and fluid, and fiber optic strands for

ES 2 218 697 T3 lighting and visualization of the blemish site. In certain embodiments of the instrument, the base of the instrument may have a studded or nail-like structure, which will help guide and position the instrument at the graft site. Of course such a nail would be designed to minimize damage to the underlying bone.

While the cutting surface of the instrument can be single tooth or multi-tooth, or describe a screw-like pattern such as a metal plug used to generate threaded holes in metal parts, the required characteristic of the cutting instrument is that the resulting sculpted sides of the graft site are wavy and non-linear. For example, in certain embodiments, the cutting edge of the instrument may be shaped similar to that shown in Figure 4A or as in Figure 4B. The cutting edge can be flat or circular in that it wraps around the diameter of the cutting instrument. Many other conformations can be designed to carry out the purpose of the method to create an interface that provides mechanical resistance to differential reaction to compression and shear forces on the transplanted material and the surrounding material.

Example 8

A four month old mixed Yorkshire pig was placed under general anesthesia and placed on its back. The pig was washed and bandaged in a surgical area at Harrington Arthritis Research Center, Phoenix, Arizona. The entire surgical procedure was carried out aseptically. The left posterior third and the adjacent abdomen and inguinal area were flushed with iodine. The knee joint was located and the patella was located. A medial incision was made, approximately 3 cm from the posterior part of the patella, and the various subcutaneous cellular tissues, muscle layers, and ligaments were cut approximately to gain access to the medial femoral epiphysis. Using a circular cutting apparatus, a lesion was prepared in the white cartilage in the medial part of the medial epiphysis, leaving a margin of 0.5 to 1 cm to the edge of the cartilage covering the posterior medial part of the epiphysis (left epiphysis , Figure 6A). The 0.5 to 1 cm imperfection was placed on a caudal weight bearing part of the medial epiphysis. The entire surgical procedure was done without a tourniquet on the left femur. The different layers and the skin were properly sutured.

On day 3 the animal was brought back to the surgical area and placed as before on the operating table and general anesthesia was given. The left posterior third, abdomen, and inguinal region were ionized as previously described. Sutures were cut and the area opened. A moderate hematoma was found to be present at the knee joint. The blood clot was removed and the blemish was inspected. There was a blood clot in the blemish that was removed. A sterile surgical instrument designed with a male threaded cutting edge sized corresponding to or slightly greater than the circumference of the lesion was carefully screwed into the imperfection. A Bio-Gide plate was cut^® to a size equal to the background of the blemish. The first glue used, called Adhesive Protein (A-2707, Sigma Chemical, USA) was applied to the dense side of the cut-out hemostatic barrier plate and the plate was placed with the dense side face down at the bottom of the lesion. , using it as a barrier as described above. It was found that this glue did not appear to dry very quickly. The slight bleeding from the bottom of the blemish stopped immediately. A second Bio-Gide® was cut somewhat larger in circumference than the lesion and placed with the dense side facing up (thus the porous side facing down towards the graft), as described above.

This non-cellular covering plate was then sutured over the cavity, leaving an open edge, where the chondrocyte to be explanted could be injected at the graft site. The surrounding portion of the edge of the plate was covered with the second glue, Dow Corning Medical Adhesive B (Cat. 895-3, Dow Corning, USA). This second glue dried much faster and more efficiently than the first glue. It was found that during this particular procedure, the first glue had not dried sufficiently to hold the hemostatic barrier in place when the lid was attempted to be sutured. The main barrier formed on the proximal surface of the graft site was by the tail itself.

Using a 1 ml syringe and a 16 gauge needle, the chondrocyte cell suspension (approximately 0.6 ml) was drawn into the barrel of the syringe. A short 23 gauge needle was exchanged for the 16 gauge needle and the cell suspension was injected under the sutured cover plate at the graft site (approximately 10 x 10⁶ cells). The open edge of the cap was then glued, prior to removal of the needle, and the needle was carefully removed. No cell leakage was seen. The wound was sutured and, as before, no tourniquet was used, no bleeding was observed. The final skin layers were sutured. No skin bulge occurred after suturing, indicating that there was no hematoma. Postoperative recovery was uneventful.

As expected, the grafted chondrocytes produced sufficient cartilage matrix to repair the prepared blemish on the articular cartilage surface of the test pig's knee joint. Figure 6A is a Nuclear Magnetic Resonance of a pig knee showing the cartilage imperfection created in the knee (left epiphysis, the medial epiphysis) and Figure 6B is a Nuclear Magnetic Resonance of the same pig knee three months after treatment showing repair of the blemish.

Example 9

A kit comprising the components useful for practicing the above method will allow timely practice on a surgical instrument. In a preferred embodiment, a kit of the invention will provide suitable sterile components for easy use in the surgical setting and will provide chondrocytes to be placed at the graft site, a suitable hemostatic barrier, suitable cover plate, and organic glue. A kit of the invention can also provide sterile, cell-free matrix material suitable for supporting autogenous chondrocytes to be implanted in a joint prosthesis surface imperfection. In one embodiment, a kit of the invention contains chondrocytes to be placed at the graft site, a Surgicel hemostatic barrier^® and a BioGide overlay plate^® with suitable coating of organic glue from Tisseel^®, where the Surgicel^® and the Bio-Gide^® they have

ES 2 218 697 T3 treated according to the explanations described above to increase the time to resorption. In cases where the Tisseel^® precoated, in one embodiment the Tisseel® is enriched with additional aprotinin to increase the time to resorption.

In another preferred embodiment, the hemostatic barrier and the cover plate are both a semi-permeable collagen matrix that is treated to extend the time to resorption of the material. It is also possible to provide Tisseel glue^® exalted as a standalone component that has to be applied as needed, due to inherent variability and the unique circumstances that will be encountered in each repair / transplant procedure.

A further embodiment of a kit of the invention will include a surgical instrument as described in Example 7 above, or various variations thereof.