Method, instruments, and kit for autologous transplantation

Field of the Invention

The present invention relates to a cartilage repair implantable device. The present invention is in the field of chondrocyte transplantation, bone and cartilage transplantation, healing, joint regeneration and prevention of joint pathologies. In particular, the invention discloses methods for preparing a transplant site, devices for such preparation, and for autologous transplantation of cells to the prepared transplant site.

State of the art

Every year in the United States of America, more than 500,000 arthroplasty and total joint replacements are performed. Approximately the same number of similar procedures are performed in Europe. These figures include around 90,000 total knee replacement procedures and around 50,000 knee defect repair procedures per year in Europe. The number of treatments is essentially the same in the United States of America (in: Praemer A., ​​Furner S., Rice, D. P., Musculoskeletal conditions in the United States, American Academy of Ortopaedic Surgeons, Park Ridge, HI., 1992, 125). A method of treating regenerative cartilage would be most useful, and could be performed at an earlier stage of joint damage, thus reducing the number of patients requiring surgery to insert an artificial joint. With such preventive treatments, the number of patients developing osteoarthritis would also be reduced.

The techniques used to restore articular cartilage surfaces have mainly attempted to induce cartilage repair using sub-cartilage drilling, abrasion, and other methods that excise diseased cartilage and sub-cartilage bone, leaving exposed vascularized spongy bone exposed (Insall, I, Clin. Ortop. 1974, 101. , 61; Ficat RP et al., Clin Ortop. 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 cartilage synthesizing cells from chicken embryo somites. Later, Cahn and Lasher (PNAS USA 1967, 58.1131) used a system to analyze the involvement of DNA synthesis as a prerequisite for cartilage differentiation. Chondrocytes respond by growth to both EFG and FGF (Gospodarowicz and Mescher, J. Cell Physiology 1977, 93, 117), but eventually lose their differentiated functions (Benya et al. Cell 1978, 15, 1313). The methods of growing chondrocytes have been described and mainly used with slight changes by Brittberg, M. et al. (New Engl. J. Med. 1994, 331, 889). Cells cultured using these methods were used as autologous grafts into the knee joints of patients. Moreover, Kolettas et al. (J. Cell Science 1995, 108, 1991) studied the expression of cartilage specific molecules such as collagens and proteoglycans under conditions of prolonged cell culture. These authors found that despite the occurrence of morphological changes during cultivation in monolayer 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), compared to suspension cultures grown on agarose gels, alginate beads or as spinner cultures (maintaining round cell morphology) tested by various scientists, markers expressed on chondrocytes such as type II collagens remained unchanged and IX, and the large proteoglycans found in aggregates, aggrecan, versican and linker protein were unchanged (Kolettas, E. et al., J. Cell Science 1995, 108, 1991).

Joint chondrocytes are specialized cells of mesenchymal origin found exclusively in cartilage. Cartilage is an avascular tissue whose physical properties depend on the extracellular matrix produced by chondrocytes. During endochondral ossification, chondrocytes mature, leading to cell hyperplasia characterized by the initiation of expression of type X collagen (Upholt, WB and Olsen, RR, In: Cartilage Molecular Aspects (Ed. Hall, B & Newman, S) CRC BocaRaton 1991, 43; Reichenberger, E. et al. Dev. Biol. 1991, 148, 562; Kirsch, T. et al. Differentiation, 1992, 52, 89; Stefens, M. et al., J. Cell Sci. 1993, 103, 1111).

Excessive degradation of type II collagen in the outer layers or articular surfaces of joints is also caused by osteoarthritis. Accordingly, the collagen network is weakened and then causes fibril formation whereby matrix substances such as proteoglycans are lost and eventually completely displaced. Such fibril formation in weakened cartilage in osteoarthritis can extend as far as calcified cartilage and into the sub-cartilage bone (Kempson, GE et al., Biochim. Biophys. Acta 1976, 428, 741; Roth, V. and Mow, VC,

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J. Bone Joint Surgery, 1980, 62A, 1102; Woo, SL-Y. et al., in Handbook of Bioengineering (R. Skalak and S. Chien eds.), McGraw-Hill, New York, 1987, pp. 4.1-4.44).

Descriptions of the basic development, histological and microscopic anatomy of bone, cartilage and other such connective tissues can be found, for example, in Wheater, Burkitt and Daniels, Functional Histology, 2nd edition, (Churchill Livingstone, London, 1987, Chapter 4). Descriptions of the basic histological anatomy of defects in bone, cartilage and other connective tissues can be found, for example, in Wheater, Burkitt, Stevens and Lowe, Basic Histopathology (Churchill Livingstone, London, 1985, Chapter 21).

Despite advances in cultivating chondrocytes and manipulating bone and cartilage, there has been little success in attempts to transplant cartilage or chondrocytes to repair damaged joint surfaces. The teachings of the present invention provide an effective and efficient means of promoting cartilage and / or chondrocyte transplantation to the site of damage at an articular junction or other cartilage-covered bone surface, whereby the cartilage is regenerated, repairing the damage. The invention also provides surgical instruments that are designed to prepare the graft site to facilitate efficient integration of the grafted material into the graft site.

The invention relates to an implantable device for the repair of cartilage by implantation in an animal, characterized in that it comprises a collagen support matrix, god cells and a biocompatible adhesive adjacent to the edge of said support matrix.

The above-mentioned god cells and the biocompatible binder are mixed homogeneously or non-homogeneously or alternately applied in one or more layers to a support matrix, said god cells and said biocompatible binder being in the form of a mixture.

Said support matrix is ​​preferably a sheet-shaped element capable of supporting the growth of the god cells and of providing the implantable device with physical integrity to facilitate its handling.

In another preferred embodiment, said carrier matrix comprises polypeptides or proteins, said collagen preferably selected from the group consisting essentially of equine, porcine, bovine, ovine and chicken collagen.

In an implantable product according to the invention, said support matrix is ​​preferably in a solid state, or alternatively such that preferably said support matrix is ​​gel-like.

In a further preferred embodiment of the invention, said collagen is type I collagen, or alternatively is type II collagen.

The implantable device of the invention is preferably reversibly deformable.

Said carrier matrix in the implantable article of the invention has a rough side. In a more preferred embodiment, the rough side is porous.

It is also possible to implement an implantable device according to the invention in which said support matrix has a smooth side, or also preferably the support matrix has a rough and smooth side.

In one embodiment, the carrier can be absorbed by the animal's body.

The implantable device for cartilage repair allows the implementation of a method of effective treatment of the cartilage covering the joint joint by chondrocyte transplantation, in a suitable matrix, on the surface to be treated, with a hemostatic barrier and a cell-free covering dressing, including: first placing a hemostatic barrier proximal to the area to be treated, placing chondrocytes in a suitable matrix on the area to be treated, distal to the hemostatic barrier, covering the area to be treated with a cell-free covering dressing. A hemostatic barrier, as will be described below, is a barrier that inhibits the penetration of cells and vascular tissue into the grafted material. In particular, the method provides a hemostatic barrier, which is an absorbable, semi-permeable material that inhibits or prevents vascular infiltration through the barrier. In one embodiment, the hemostatic barrier comprises collagen. The term cell-free is used herein as is used in the art to denote material that is substantially free of intact cells that are capable of subsequent cell division, expansion, or biological activity. The cell-free material may be free of any intact nucleated cells. In one embodiment, the present method comprises the use of a cell-free cover dressing that includes a semipermeable collagen matrix. In one variation of the treatment method, the porous surface of the acellular covering dressing faces the implant material.

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Also disclosed herein is autologous transplantation of collagen or chondrocytes to a graft site where the graft site was first made by surgical manipulation for better acceptance of the grafted material. The graft site should be carved so that the walls of the graft site are shaped in an undulating manner so that the grafted material, when placed in the graft site and unfolded so that it comes into contact with the wall of the graft site, is resistant to removal or expulsion of the entire graft from the site. transplant. This autologous collagen transplant is performed with surgical instruments designed to carve the graft site.

For the transplantation of cartilage and / or chondrocytes onto the joint surface, a kit comprising a hemostatic barrier, a cell-free semi-permeable covering dressing and an organic adhesive are useful. The kit can optionally further include one or more surgical instruments that can be used to carve the graft site according to the methods set forth herein.

Brief description of the drawing

The subject of the invention in the exemplary embodiments is shown in the drawing in which:

Fig. 1A shows a typical articular bone end. Typically, the bone material is covered on the articular surface with a god-cap (indicated by hatching).

Fig. 1B shows an example of a defect or damage to the godock (hatch gap), and such damage can be directly treated, slightly enlarged or carved for attachment of the grafted material by surgical means prior to treatment.

Fig. 1C shows how a hemostatic barrier (blackened area, indicated by 1) is placed within the lesion and in the godock to inhibit or prevent vascularization of the regenerating cartilage from the underlying bone. The chondrocytes to be implanted into the defect cavity are then layered on top of the hemostatic barrier.

Fig. 2 is a drawing showing a treated defect (gap in the hatched area) in the godock (hatched area) covered with a cell-free semi-permeable material (blackened area, indicated by 2) that is used to form a cap / dressing or bandage over the defect site. This cap is either fixed in place or sewn to the edge of the cavity onto healthy cartilage or attached in some other way. This cap covers the surface of the joint defect where the glue and the cultured chondrocyte / cartilage graft have been placed.

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

Fig. 3B illustrates the graft site after the defect has been carved to have wavy walls.

Fig. 3C illustrates a carved graft site with a hemostatic barrier 1, grafted material 3, and a cell-free covering dressing 2 in place within the cartilage of the articular surface 4.

Fig. 4A illustrates one embodiment of the surgical instrument showing cutter teeth 5 and protruding locating pin 6.

Fig. 43 illustrates a second embodiment of the surgical instrument.

Fig. 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.

Fig. 6A is an MRI of a pig knee showing a cartilage defect in the left (medial) condyle.

Fig. 6B is an MRI of the same pig knee three months after treatment.

Fig. 7 illustrates a graft site or defect 23 without a hemostatic barrier, including a mixture of transplanted cells 21 and a biocompatible adhesive 22 on a support matrix 18 having a porous side, baptism material and graft material interface 30, cartilage 10, bone and berry material interface 12. , absorbable pin 24 and bone material 14.

Detailed Description of the Invention

The invention relates to an implantable device for cartilage repair, the article containing products that inhibit the formation of vascular tissue, such as, for example, capillary loops to grow into cartilage formation during the autologous transplantation process of chondrocytes into cartilage defects. The formation of vascular tissue from the underlying bone will be

Tend to project into new cartilage to form, leading to the emergence of cells other than the desired mesenchymal specialized chondrocytes.

Contaminating cells introduced by vascularization can initiate the entry and overgrowth of implanted chondrocytes into the cartilage to be formed. One type of commercial product that can be used in the product of the invention is Surgicel® (Ethicon Ltd., UK) which can be absorbed after a period of 7-14 days. The use of this substance in the method of the invention is contrary to the normal use of a hemostatic device such as the Surgicel® as described in the insert from Ethicon Ltd.

It has surprisingly been found that when it is desired to inhibit cartilage re-vascularization, the hemostatic material will act as a gel-like artificial coagulum. If red blood cells emerge within the full-thickness defect of articular cartilage covered by such a hemostatic barrier, these red blood cells will be chemically changed to hematin, and thus unable to induce vascular growth. Thus, a hemostatic product when used as a barrier to inhibit re-vascularization with or without fibrin binders, such as, e.g., Surgicel®, is effective in the present invention. As a dressing to cover the surface of the joint defect, a cell-free component may be used into which cultured chondrocytes / cartilage are transplanted using autologous chondrocytes for transplantation. The invention also contemplates the use of suitable allogeneic chondrocytes or xenogeneic chondrocytes for the repair of a cartilage defect.

Thus, the present invention allows for the effective repair or treatment of cartilage defects in the bone surfaces forming the articular junction that includes administering an agent or device for blocking vascular invasion to the cartilage site to be repaired, and providing an acellular barrier that will isolate the site. repair and keep transplanted cells in place. The present invention also provides a kit comprising a hemostatic barrier component for insertion into the site to be repaired such that there is effective inhibition of vascularization in the site to be repaired; and once the chondrocytes to be transplanted have been placed on the site to be repaired, the repair site is covered with an acellular semi-permeable barrier so that the transplanted chondrocytes are kept in place but still have access to nutrients.

Certain aspects of the invention are exemplified by using an in vitro system to study the behavior of chondrocytes in contact with a certain product or combination of certain products that inhibit the formation of vascular tissue. This in vitro testing predicts the ability of certain test materials to inhibit vascularization, such as occurs in vivo where the capillary loops grow towards the cartilage formed during the autologous chondrocyte transplant process into cartilage defects.

Suitable hemostatic products will have the ability to inhibit the growth or invasion of vascular tissue, osteocytes, fibroblasts, etc. into developing cartilage. Appropriate hemostatic material should prevent vessel and cell invasion into the developing cartilage in order to optimize cartilage formation and achieve full thickness repair of any articular cartilage defects. Ideally, the hemostatic barrier will last for an extended period of time sufficient to allow the cartilage to fully repair and then be able to be resorbed or otherwise degraded over time. One material identified as suitable is called Surgicel® W1912 (an absorbable hemostat containing oxidized regenerated sterile cellulose; Lot GG3DH, Ethicon Ltd. UK). Another example of a suitable material is BioGide® (a commercially available type I collagen matrix dressing; Geistlich Sohne, Switzerland).

A suitable organic adhesive can be found on the market, such as, e.g., Tisseel® or Tissucol® (fibrin-based adhesive; Immuno AG, Austria), Adhesive Protein (Cat. # A-2707, Sigma Chemical, USA), and Dow Corning Medical Adhesive B (Cat. # 895-3, Dow Corning, USA).

Surgical instruments contemplated by the present invention can be manufactured from metal and / or plastic suitable for making disposable or reusable surgical instruments. The cutting jig may include cutting teeth that are completely round or flat, or any shape between the two. Since cartilage is a relatively soft material, it may be advantageous to produce hardened plastic cutting edges that can sculpt the cartilage without damaging the bone. Such cutting devices may be manufactured to include openings for fluid delivery, suction of scraps and fluids, and optical fibers for illumination and visualization of the defect site.

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In the present invention, the implantable device comprises cells 21 and adhesive 22 joined together as (1) a mixture of adhesive 22 and cells 21, or (2) one or more alternating layers of cells 21 and adhesive 22 on the carrier matrix 18.

In one embodiment, the cells 21 are autologous cartilage cells to be transplanted into the lesion 23. The chondrocytes 21 are mixed, homogeneously or non-homogeneously, with the appropriate adhesive 22 before applying the chondrocyte / adhesive mixture to the support matrix 18. Preferably, the adhesive 22 and 21 chondrocytes are mixed directly (i.e. in an operating room) before applying adhesive 22 and cells 21 to support matrix 18, and implanting a combination of adhesive 22, cells 21 and support matrix 18 into the defect 23. Alternatively, the cells 21 and adhesive 22 are alternately applied in one or more layers to support matrix 18 In one embodiment, glue 22 is a biocompatible glue, such as a fibrin glue, more specifically either an autologous fibrin glue or a non-autologous fibrin glue. Preferably, an autologous fibrin glue is used.

Once the chondrocytes 21 are bonded to the adhesive 22 on the support matrix 23, the adhesive / chondrocyte / support matrix combination is implanted into the defect 23 with or without a hemostatic barrier (as described herein) between bone 14 and the adhesive / chondrocyte / support matrix combination. In one embodiment, as shown in Fig. 2, the defect 23 treated does not contain a hemostatic barrier between bone 14 and the adhesive / chondrocyte / carrier matrix combination.

In one embodiment, shown in Fig. 7, treatment of defect 23 is similarly performed without the use of a hemostatic barrier between the adhesive / chondrocyte / carrier matrix combination and the defect base 23.

Further, in the embodiment shown in Fig. 7, the combination of adhesive 22 and chrondrocytes 21 is only present on a portion of the support matrix 18. For example, the adhesive 22 and chondrocytes 21 are preferably only on the outer edge of the porous support matrix 18 side. Alternatively, chondrocytes 21 and / or the adhesive 22 penetrates the porous side of the support matrix 18 to a desired level, e.g. 2 to 50% of the depth of the support matrix 18. Typically, the porosity of support matrix 18 is greatest closest to the rough side, i.e. the side where the cells 21 and adhesive 22 are deposited, and smallest on the opposite side of support matrix 18. As further illustrated by Fig. 7, the porosity of support matrix 18 is indicated by shading. where darker shades indicate less porosity and lighter shades indicate greater porosity.

In yet another embodiment, chondrocytes 21 are applied to the support matrix 18, then coated with a suitable biocompatible adhesive 22 layer. Although a single layer of chondrocytes 21 on support matrix 18 and a single layer of adhesive 22 applied to chondrocytes 21 is sufficient to effectively treat the defect, more can be used. than one alternating layer of glue 22 and chondrocytes 21. Alternatively, the biocompatible adhesive layer 22 may be initially deposited on the support matrix 18 and then the chondrocyte layer 21 may be deposited on the adhesive 22.

In one embodiment, the support matrix 18 is a sheet-shaped member capable of supporting the growth of chondrocytes 21 and providing physical integrity to the implantable device to facilitate handling. In one embodiment, the support matrix 18 comprises polypeptides or proteins such as collagen. In particular, carrier matrix 18 includes equine, porcine, bovine, ovine and chicken collagen. Moreover, the collagen can be either type I or type II. Examples of suitable collagen carrier matrices 18 include ChondraGide® and BioGide® (commercially available type I collagen matrix dressings; from Geistlich Sohne, Switzerland). Moreover, the support matrix 18 is reversibly deformable and has either a rough, ie porous side, as described above, or a smooth side, or two rough sides. Although either side can be porous, in one embodiment as described herein, the rough side is more porous than the smooth side. The carrier matrix 18 and the cells 21 thereon can optionally be held in place in the defect with additional biocompatible adhesive 22 and / or one or more biocompatible absorbable pins 24 as shown in Fig. 7. In one embodiment, one or more aliquots of carrier matrix 18 have gel-like or solid properties.

The invention has been illustrated on the basis of the following examples.

Example 1

In order to use the Surgicel® in the product of the invention to prevent the growth of blood vessels into autologous implanted cartilage or chondrocytes, the Surgicel® was first treated with a fixative such as glutaraldehyde. Briefly, the Surgicel® was treated with 0.6% glutaraldehyde for 1 minute and then washed several times to eliminate residual glutaraldehyde that could otherwise be toxic to the tissue. Alternatively, the Surgicel® was treated with a fibrin sealant called Tisseel® prior to treatment with glutaraldehyde as described in Example 2. Surgicel® was found to be fixed with, for example, a fixative such as glutaraldehyde, washed with sterile saline (0.9%) and stored in a refrigerator , it does not dissolve for 1 to 2 months. Generally, Surgicel® is resorbed between 7 and 14 days. This time would be too short as longer time is needed to prevent blood vessel growth or vascularization as such from the bone structure to the implanted cartilage before the implanted chondrocytes transform into a layer of solid cartilage to meet their nutritional needs from adjacent cartilage. In other words, sufficient vascularization inhibition is needed over a longer period of time, such as for example one month. Thus, the product should not be absorbed much sooner. On the other hand, resorption is needed last. Thus, the organic material used as the inhibitory barrier will have these properties, and Surgicel® treated in this manner has been found to provide this function.

Example 2

Surgicel® was also coated with an organic adhesive, in this example the adhesive used was Tisseel® but others could also be used. This product, together with Surgicel®, produces a usable barrier for a specific purpose. Any other hemostat or vascular inhibitory barrier can be used. The Tisseel® was mixed as described below. The Surgicel® was then coated with Tisseel® adhesive by spraying the Surgicel® material on both sides until soaked.

The Tisseel® (fibrin glue) was then allowed to solidify at room temperature.

Immediately before solidification was complete, the coated Surgicel® was placed in 0.6% glutaraldehyde for 1 minute and then washed with sterile saline (0.9%). The pH was then adjusted by adding PBS and / or NaOH until the pH was adjusted to 7.2 to 7.4. Thereafter, the treated Surgicel® was washed in tissue culture medium such as minimally concentrated stock / F12 medium with 15 mM Hepes buffer.

As mentioned in this example, Tisseel® was used as a fibrin sealant to coat the Surgicel®. In addition, a fibrin sealant or glue can also be applied directly to the bottom of the lesion towards the bone to which the Surgicel® is adhered. The in vitro system used, instead of in vivo testing, consisted of a sterile disposable 6-well MJNCLON plate^TM Delta for cell research (NUNC. InterMed, Roskilde, Denmark). Each well is approximately 4 cm in diameter.

According to the invention, the fibrin sealant may be any adhesive that together with the fibrin component will form an adhesive that can be tolerated in humans (Biara, N, et al., Burns Incl. Therm. Inj., 1984, 10, 396). The invention also contemplates any other adhesive that can be used in place of the fibrin sealant. According to this invention, Tisseel® or Tissucol® (Immuno AG, Vienna, Austria) was used. The Tisseel® kit consists of the following components:

Tisseel, a lyophilized, virus-inactivated sealant, comprising the clotting protein: fibrinogen, plasma fibronectin (CIG) and factor XIII, and plasminogen;

- Aprotinin solution (bovine);

- Thrombin 4 (bovine);

- Thrombin 500 (bovine); and

- calcium chloride solution.

The Tisseel® Kit includes the DUPLOJECT® Application System. A fibrin sealant or two-component sealant using the Tisseel® Kit is bonded as follows according to the Immuno AG product insert.

Example 3

Chondrocytes were grown in a minimal amount of HAM F12 stock culture medium containing 15 mM Hepes buffer and 5 to 7.5% autologous serum in a CO2 incubator at 37 ° C and made in a Class 100 laboratory at Verigen Europe A / S, Symbion Science Park, Copenhagen, Denmark. Other culture medium compositions can be used to cultivate chondrocytes. Cells were trypsinized with trypsin EDTA for 5 to 10 minutes and counted by viability staining with Trypan Blue in a Burker-Turk chamber. The number of cells was adjusted to 7.5x10⁵ cells per ml. One NUNCLON plate^TM was unveiled at the Class 100 Laboratory.

The Surgicel® hemostatic barrier was cut to size to fit the bottom of the well in the NUNCLON ™ tissue culture tray. In this case, a circle approximately 4 cm in size (but could be of any possible size) was cut and placed under asep8 conditions.

On the bottom of the well in a sterile disposable NUNCLON 6-well plate^TM Delta for cell research (NUNC, InterMed, Roskilde, Denmark). A hemostatic barrier prepared to be placed at the bottom of the well was pretreated as described in Example 1. This treatment significantly delays the absorption of the Surgicel. This hemostatic barrier was then washed several times in distilled water and then several times until unreacted glutaraldehyde was washed away. A small amount of serum-containing cell culture medium was used in order to be absorbed by the hemostatic barrier and, at the same time, to maintain the hemostatic barrier moist at the bottom of the well.

Approximately 10⁶ cells in 1 ml of culture medium were placed directly on top of the hemostatic barrier, spread over the surface of the hemostatic barrier pretreated with 0.4% glutaraldehyde as described above. The plate was then incubated in a CO2 incubator at 37 ° C for 60 minutes. Tissue culture medium in an amount of 2 to 5 ml containing 5 to 7.5% serum was carefully added to the well containing the cells, avoiding cell splashing by keeping the pipette tip tangential to the side of the well while draining the medium. It turned out that the pH of the substrate was too low (pH ~ 6.8). The pH was then adjusted to 7.4 to 7.5. The next day, some chondrocytes began to grow in clusters on the hemostatic barrier. Certain cells died due to exposure to low pH prior to its regulation. The plate was incubated for 3 to 7 days with the medium changed on day 3.

At the end of the incubation period, the medium was decanted, and chilled 2.5% glutaraldehyde containing 0.1M sodium dimethyl arsenic acid, (also called sodium cacodylate, pH adjusted with HCl to 7.4) was added as a fixative to prepare the cell and carrier ( haemostatic barrier) for subsequent preparation for electron microscopy.

Example 4

Chondrocytes were grown in a minimal amount of HAM F12 stock culture medium containing 15 mM Hepes buffer and 5 to 7.5% autologous serum in a CO2 incubator at 37 ° C and made in a Class 100 laboratory at Verigen Europe A / S, Symbion Science Park, Copenhagen, Denmark. Other culture medium compositions can be used to cultivate chondrocytes. Cells were trypsinized with EDTA for 5 to 10 minutes and counted by viability staining with Trypan Blue in a Burker-Turk chamber. The number of cells was adjusted to 7.5x10⁵ cells per ml. One NUNCLON plate^TM was unveiled at the Class 100 Laboratory.

Surgicel® (for use as a hemostatic barrier) was treated with 0.6% glutaraldehyde for one minute as described in Example 1, and washed with 0.9% sterile sodium chloride solution or preferably a buffer such as PBS buffer or a culture medium such as MEM / F12 because the pH after treatment with glutaraldehyde is 6.8 and should preferably be 7.0 to 7.5. Tisseel® was applied to both sides of the Surgicel® using the DUPLOJECT® system, thereby covering both sides of the Surgicel®, the dressing to be applied, with fibrin sealant. The adhesive is allowed to dry under aseptic conditions for at least 3 to 5 minutes. "The coated hemostatic barrier was placed at the bottom of the well in a sterile disposable NUNCLON 6-well plate.^TM Delta for research on cells. A small amount of serum-containing tissue culture medium was used to absorb it by the hemostatic barrier. Approximately 10⁶ cells in 1 ml serum-containing tissue culture medium were placed directly on top of the Hemostat, dispersed over the surface of the hemostatic barrier. The plate was then incubated in a CO2 incubator at 37 ° C for 60 minutes. 2 to 5 ml of tissue culture medium containing 5 to 7.5% serum was carefully added to the well containing the cells avoiding cell splashing by keeping the pipette tip tangential to the side of the well while draining the medium. After 3 to 6 days, microscopic examination showed that cells adhered to and grew into the Surgicel® satisfactorily, suggesting that the Surgicel® was not toxic to chondrocytes and that chondrocytes grew satisfactorily in the Surgicel®.

The plate was incubated for 3 to 7 days by changing the medium on day 3. At the end of the incubation period, the medium was decanted and cooled, and 2.5% glutaraldehyde containing 0.1M sodium dimethyl arsenic acid, also called sodium cacodylate, pH adjusted with HCl was added to 7.4, as a fixative for the preparation of a cell and a carrier (hemostatic barrier) for subsequent preparation for electron microscopy.

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Example 5

Chondrocytes were grown in a minimal amount of HAM F12 stock culture medium containing 15 mM Hepes buffer and 5 to 7.5% autologous serum in a CO2 incubator at 37 ° C and made in a Class 100 laboratory at Verigen Europe A / S, Symbion Science Park, Copenhagen, Denmark. Cells were trypsinized with EDTA for 5 to 10 minutes and counted by viability staining with Trypan Blue in a Burker-Turk chamber. The number of cells was adjusted to 7.5x10⁵ up to 2x10⁶ cells per ml. One NUNCLON plate^TM was unveiled at the Class 100 Laboratory.

It has been found that Bio-Gide® can be used as an absorbable bilayer membrane that will be used as a dressing or bandage covering the surface of the defect in the joint into which cultured chondrocytes are transplanted as well as the hemostatic barrier. Bio-Gide® is a pure collagen membrane obtained by standardized, controlled manufacturing processes (ED Geistlich Sohne AG, CH-6110 Wolhusen). Collagen is obtained from veterinary certified pigs and carefully purified to avoid antigenic reactions and sterilized in double blisters with gamma irradiation. The two-layer film has a porous surface and a dense surface. The membrane is made of type I and type III collagen without any subsequent cross-linking or chemical treatment. Collagen is resorbed within 24 weeks. The membrane maintains its structural integrity even when wet and can be secured with stitches or nails. The membrane may also "adhere using a fibrin sealant such as Tisseel® to adjacent cartilage or tissue, either in place of sutures or simultaneously with sutures.

Bio-Gide® was unveiled in a Class 100 Laboratory and placed aseptically at the bottom of the well in a sterile disposable NUNCLON 6-well plate^TM Delta for cell research by facing up a porous surface or a dense surface of a two-layer membrane. Approximately 10⁶ cells in 1 ml serum-containing tissue culture medium were placed directly on top of the Bio-Gide®, scattering either over the porous or compact surface of the Bio-Gide®. The plate was then incubated in a CO2 incubator at 37 ° C for 60 minutes. Tissue culture medium in an amount of 2 to 5 ml, containing 5 to 7.5% serum, was carefully added to the well containing the cells avoiding cell splashing by keeping the pipette tip tangential to the side of the well while draining the medium.

On day 2, after the chondrocytes were placed in the Bio-Gide® well, the cells were examined under a Nikon inverted microscope. Certain chondrocytes were found to adhere to the edge of BioGide. Obviously, it was not possible to see Bio-Gide® alone using this microscope.

The plate was incubated for 3 to 7 days by changing the medium on day 3. At the end of the incubation period, the medium was decanted and chilled 2.5% glutaraldehyde containing 0.1 M sodium dimethyl arsenic acid, also called sodium cacodylate, pH adjusted with HCl to 7 was added , 4, as a fixative for preparing a cell and Bio-Gide® carrier with cells grown on a porous surface or a compact surface. The Bio-Gide® dressings were then sent for electron microscopy to the Department of Pathology, Herlev Hospital, Denmark.

Electron microscopy showed that chondrocytes grown on the compact surface of Bio-Gide® did not grow into the collagen structure of Bio-Gide®, while cells grown on the porous surface actually grew into the collagen structure and, furthermore, showed the presence of proteoglycans and no signs of fibroblast structures. This result indicates that when a collagen dressing such as a Bio-Gide® dressing is sutured to cover a cartilage defect, the porous surface will face the defect into which cultured chondrocytes are to be injected. They will then be able to penetrate the collagen and produce a smooth cartilage surface in line with the intact surface, and a smooth layer of proteoglycans will be built up in this area. Whilst, if the dense surface of the collagen is facing down into the defect, the chondrocytes to be implanted will not integrate into the collagen and the cells will not produce the same smooth surface as described above.

Example 6

Chondrocytes were cultured in a minimal amount of the basic culture medium containing HAM F12 and 15 mN Hepes buffer and 5 to 7.5% autologous serum in a CO2 incubator at 37 ° C and made in the Class 100 laboratory at Verigen Europe A / S, Symbion Science Park, Copenhagen, Denmark. Cells were trypsinized with EDTA for 5 to 10 minutes and counted via viability staining with Trypan Blue in a Burker-Turk chamber. A number

Cells were adjusted to 7.5x10⁶ up to 2x10⁶ cells per ml. One NUNCLON plate^TM was unveiled at the Class 100 Laboratory.

Bio-Gide® when used as an absorbable bilayer film can also be used together with an organic adhesive such as Tisseel® with an additional, much higher content of Aprotinin than is normally found in Tisseel®, as described in the product insert. By increasing the Aprotinin content to about 25,000 KTU / ml, the resorption of the material will be delayed by weeks and instead of the normal period of days.

To test this feature in vitro, Tisseel® is applied to the bottom of the well of the NUNCLON plate.^TMand it is left to solidify incompletely. A collagen dressing such as Bio-Gide® is then applied over the Tisseel® and glued to the bottom of the well. This combination of Bio-Gide® and Tisseel® is designed as a hemostatic barrier that will inhibit or prevent the development or infiltration of blood vessels into the chondrocyte transplant area. This hybrid collagen dressing can now be used both as a hemostatic barrier at the bottom of the lesion (closest to the area to be repaired) as well as a vehicle for cartilage formation, since the distal surface can be the porous side of the collagen dressing and thus aid infiltration of chondrocytes and cartilage matrix. Thus, this hybrid collagen dressing can also be used to cover the top of an implant with a collagen porous surface facing downwards towards the implanted chondrocytes and a barrier forming the top. The hybrid collagen dressing, with a higher content of Aprotinin, can also be used without any organic adhesive such as Tisseel® and placed directly into the cavity, adhering due to natural forces. Thus, the collagen dressing can be used both as a hemostatic barrier and as a cell-free covering of the repair / graft site, with the porous surfaces of the dressings facing towards the grafted chondrocytes / cartilage. In another embodiment, a collagen dressing that consists of type II collagen (i.e. from Geistlich Sohne AG, CH-6110 and Wolhusen) would be used.

Thus, the object of the present invention is a hybrid collagen dressing which is a collagen matrix dressing with an increased Aprotinin content, preferably about 25,000 KlU / ml, combined with an organic matrix adhesive, the collagen component being similar to the absorbable bi-layer material of Bio-Gide. ® or type II collagen, and the organic adhesive is similar to Tisseel®. In another embodiment, the hybrid collagen dressing does not use any organic adhesive to adhere to the repair site.

Example 7

Due to the weakened cartilage structure in osteoarthritis, the adhesion of cultured autologous chondrocytes transplanted into the graft site in defective cartilage may be inhibited, thus creating a marginal zone (demarcation zone) between recently implanted cartilage / chondrocytes and the surrounding fixed cartilage. This marginal zone will be most pronounced if the graft site is prepared for grafting by creating straight, smooth linear cut walls. Shear and compressive forces across such a marginal zone (as illustrated in Fig. 3A) will exert great graft displacement force when the graft site is cut linearly. This marginal zone, and the differential movement of materials along this zone, will inhibit healing by the coalescence of the grafted material and the surrounding material. In many cases, the grafted material is softer than the surrounding material, however, in some cases of osteoarthritis, the surrounding cartilage may actually be softer than the implanted chondrocytes / cartilage.

Thus, to solve this problem, the method of the invention teaches the use of surgical instruments for sculpting the walls of the graft site so that the walls are non-linear and thus provide undulating surfaces. It is also possible to shape the graft site so that the diameter of the site proximal to the bone surface is greater than the opening distal to the bone and near the cartilage surface. However, a preferred embodiment describes sculpting the walls of the graft site in a manner similar to a threaded hole for a screw or screw (as illustrated in Fig. 3B), thus providing mechanical resistance to compression and / or pushing of the grafted material out of the graft site, which can be described as male and female thread.

Surgical instruments contemplated by the present invention can be manufactured from metal and / or plastic suitable for making disposable or reusable surgical instruments. Since cartilage is a relatively soft material, it can be advantageous to produce hardened plastic cutting edges that can sculpt the cartilage without being able to

Bone damage. Such cutting instruments may be fabricated to include openings for fluid delivery, for sucking up scraps and fluid, and optical fibers for illuminating and visualizing the defect site. In some implementations of the device, the base of the device may have a protruding point or pin-like structure that will aid in guiding and positioning the device at the site of a transplant. Obviously, such a pin would be designed to minimize damage to the underlying bone.

While the cutter surface of the instrument may be single serrated, or multiple serrations, or characterized by a screw pattern such as in a metal tap used to create threaded holes in metal parts, the property of the cutter is required to ensure that the resulting sculpted sides of the graft site are wavy and nonlinear. For example, in some embodiments, the cutting edge of the tool can be shaped similar to the shape shown in Fig. 4A or in Fig. 4B. The cutting edge may be flat or circular in that it is wrapped around the diameter of the cutting device. Many other shapes can be designed to achieve the object of the invention to create an interface that provides mechanical resistance to the differential response to compressive and shear forces on the graft and the surrounding material.

Example 8

A four-month-old Yorkshire hybrid pig was given general anesthesia and placed on its back. The pig was washed and dressed for surgery in the operating room of the Harrington Arthritis Research Center, Phoenix, Arizona. The entire surgical procedure was performed aseptically. The left hind leg and adjacent abdominal and groin areas were iodine cleaned. The knee joint and the kneecap are located. A medial incision was made approximately 3 cm from the back of the patella and cut several layers of subcutaneous tissue, muscles and ligaments nearby for access to the medial femoral condyle. Using a circular knife, a lesion was created in the white cartilage on the medial part of the medial condyle, leaving a margin of 0.5 to 1 cm from the edge of the cartilage covering the posterior-medial part of the condyle (left condyle. Fig. 6A). A defect of 0.5 to 1 cm was placed in the caudal mass supporting part of the medial condyle. The entire surgical procedure was performed without a tourniquet on the left thigh. The various layers and the skin were properly sewn together.

On day 3, the animal was brought back to the operating room and placed on the operating table as above and given general anesthesia. The left hind leg and the adjacent abdominal and groin area were disinfected with iodine as described above. The seams were cut open and the area was opened. A moderate hematoma was noted in the knee joint. The blood clot was removed and the defect was examined. There was a blood clot in the defect that I will remove this.

A sterile surgical instrument designed with a male threaded cutting edge sized to fit or slightly larger than the circumference of the lesion and carefully threaded into the defect. BioGide® dressing was cut to a size equal to the size of the cavity floor. The first adhesive used, Adhesive Protein (A-2707, Sigma Chemical, USA) was applied to the dense side of the cut hemostatic barrier dressing, and the dressing was placed with the dense side down on the bottom of the lesion using it as a barrier as described above. It was found that this adhesive did not dry very quickly. Slight bleeding from the bottom of the defect was stopped immediately. The second BioGide® was trimmed to have a slightly larger circumference than the lesion and placed with the compact side up (thus the porous side down towards the graft) as described above.

This non-cellular covering dressing was then sutured to the cavity, leaving one edge open where the grafted chondrocytes could be injected. The surrounding portion of the edge of the dressing was coated with a second adhesive, Dow Corning Medical Adhesive B (Cat. # 895-3, Dow Corning, USA). The latter glue was dried much faster and more efficiently than the first glue. During this particular procedure, it was found that the first glue did not dry sufficiently to hold the hemostatic barrier in place when the cap was sewn on. Thus, the main barrier on the proximal surface of the graft site was formed by the adhesive itself.

Using a 1 ml syringe and a 16 gauge needle, the chondrocyte suspension (approximately 0.6 ml) was drawn into the syringe barrel. A 23 gauge short needle was changed to a 16 gauge needle, and the cell suspension was injected under the sutured covering dressing into the graft site (approximately 10x10⁶ cells). The open edge of the cap was then taped before the needle was removed, and the needle was carefully withdrawn. No cell leakage was observed. The wound was sutured and, as above, no tourniquet was used, no bleeding was observed. The final layers of skin were sewn together. After suturing, the skin did not protrude, indicating that there was no hematoma. Recovery after surgery was uneventful.

PL 210 783 B1

As expected, the transplanted chondrocytes produced a cartilage matrix sufficient to repair a defect made in the articular cartilage surface of the test pig's knee joint. Fig. 6A is an MRI of a pig knee showing a cartilage defect formed in the knee (left condyle, medial condyle), and Fig. 6B is an MRI of the same pig knee three months after treatment showing defect repair.

Example 9

A kit containing the components useful for practicing the method according to the invention will enable convenient application of the method according to the invention in a surgical setting. In a preferred embodiment, the kit of the invention will provide sterile ingredients suitable for easy application in a surgical environment, and will provide a suitable hemostatic barrier, a suitable covering dressing, and if necessary an organic adhesive. The kit of the invention may also provide a sterile, cell-free matrix material suitable for supporting autologous chondrocytes to be implanted into an articular joint surface defect. In one embodiment, the kit of the invention comprises a Surgicel® hemostatic barrier and a Bio-Gide® overlay dressing with a suitable coating in the form of the Tisseel® organic adhesive, where the Surgicel® and Bio-Gide® have been treated according to the indications of the invention to increase the time to elapsed. resorption. In cases where Tisseel® is pre-applied, in one solution Tisseel® is supplemented with additional aprotinin to increase the time to resorption.

In another preferred embodiment, both the hemostatic barrier and the covering dressing are a semipermeable collagen matrix that is treated to increase the time for the material to resorb. It is also possible to supply the Tisseel® adhesive in a reinforced form as a separate component for use as needed due to the inherent variability and the exceptional circumstances that the repair / transplant procedure will encounter.

Further, the kit design will include a surgical instrument as described above in Example 7.

It should be understood by those skilled in the art that numerous changes and / or modifications can be made to the invention provided in specific embodiments without departing from the spirit and scope of the invention as broadly described herein.