WO2005079881A1

WO2005079881A1 - Arthroscopic method for cell implantation in mammals

Info

Publication number: WO2005079881A1
Application number: PCT/DK2005/000107
Authority: WO
Inventors: Peter Storgaard
Original assignee: Interface Biotech A/S
Priority date: 2004-02-19
Filing date: 2005-02-18
Publication date: 2005-09-01

Abstract

The present invention relates to an endoscopic method for treating cartilage or bone defects in a mammal, said method comprising the steps of: i.-dentifying the position of the defect, ii.-applying a cell suspension comprising cells selected from the group consisting of chondrocytes, chondroblasts, osteocytes and osteoblasts and combinations thereof into the cartilage or bone defect, the defect being positioned in such a manner that the cell suspension substantially does not flow out of the defect upon application.

Description

Arthroscopic method for cell implantation in mammals

Headline.

Arthroscopic Autologous Cell Implantation in articular joints of mammals, explant cultured chondrocytes/chondroblasts and/or osteocytes/osteoblasts, scope, catheter, gravitational force, sedimentation and cation-dependent cell adhesion.

Field of the invention

To enhance the repair of cartilage and bone defects in small (e.g., finger, toes, spine) and large (e.g., elbow, knee, hip) joints, an Arthroscopic Autologous Cell Implantation method has been designed. The method enables the orthopedic surgeon to perform arthroscopic implantation of explant-cultured cells, without open joint surgery in mammals.

Background of the invention

Autologous Chondrocyte Implantation (ACI). Articular cartilage has a limited capacity for repair after injury. To enhance the repair of articular cartilage defects, Autologous Chondrocyte Implantation (ACI) has been introduced with great clinical results.

ACI is a clinical procedure for articular cartilage repair of the knee joint. In clinical studies and animal experiments, cartilage defects have been treated by cultured chondrocytes which are injected under a periosteal flap. These chondrocytes are retained within the defect, and begin to synthesize matrix components to fill the defect. The newly formed matrix integrates with the surrounding host hyaline cartilage and after a certain time, solid repair tissue ("hyaline-like" cartilage) is formed in the defect.

As the current ACI method requires open joint surgery and suturing of a periosteal flap/patch to cover the injected chondrocytes, the surgery method is labor intensive. Furthermore, the current ACI procedure gives the patients complications related to healing of the shine bone after harvesting of the periosteal flap as well as healing of the operated knee after open knee surgery. Other complications related to ACI are graft hypertrophy and detachment.

A new arthroscopic ACI method, which eliminates open knee surgery, harvesting of the periosteal flap on the shinbone and the suturing of the periosteal flap to the cartilage defect, is highly warranted. Additionally, it is desirable to be able to use the ACI technology in small joints.

The following invention describes such a method, wherein articular cartilage and/or bone are repaired with cultured autologous chondroblast chondrocytes or osteoblast/osteocytes, which are arthroscopically implanted into a defect.

Description of the invention

The objective of the invention is to provide an arthroscopic ACI treatment method of articular cartilage or bone defects in various joints in the human body or in animals. The method provides a technique wherein a cell suspension is applied through a portal ("keyhole") into the articular joint, leading to sedimentation of cultured cells and adhesion of cells to subchondral bone and/or cartilage. The present invention therefore relates to an endoscopic method for treating cartilage or bone defects in a subject, said method comprising the steps of: i. identifying the position of the defect, ii. applying a cell suspension comprising cells selected from the group consisting of chondrocytes, chondroblasts, osteocytes and osteoblasts and combinations thereof into the cartilage or bone defect, the defect being positioned in such a manner that the cell suspension substantially does not flow out of the defect upon application.

When an arthroscope is used to identify the position of the defect in step i), and used to guide the injection of cultured cells in step ii), the method is known as Arthroscopic ACI.

In the context of the present application and invention the following definitions apply:

Arthroscopic Autologous Cell Implantation (arthroscopic ACI) is a medical procedure for treating cartilage or bone defects, whereby cultured cells are implanted into a defect by a catheter. This implantation procedure is visualized and guided by an arthroscope. The Cell Adhesion Buffer \s intended to mean a solution that resists changes in pH and which supports an ion dependent adhesion of chondrocytes to cartilage and bone after arthroscopic implantation. It forms the liquid phase of the cell suspension which is applied to the defect. It is usually an aqueous medium, and allows the cells to be applied to the defect via a catheter or syringe while maintaining them under suitable conditions. The "Cell Adhesion Buffer" contains divalent metal-ions such as Ca²⁺, Mg²⁺, Mn²⁺ which, are essential for obtaining binding between cell adhesion molecules on the cells and ligands in the matrix. Various buffer systems can be used during delivery of cartilage and bone cells to the defect area. Standard buffers system used in relation to this invention are; Phosphate Buffered Saline (PBS), cell culture medium (DMEM, M 199) or medium containing human (HS) or animal serum (FCS).

A catheter is a flexible tube, which is inserted into the body of the subject and used for introducing or removing material. In this application, the catheter is used to inject cells into the defect under treatment. During arthroscopic autologous chondrocyte implantation, the catheter is either anchored directly to an arthroscope or the catheter is used alone for injection of cells into the articular joint. Alternatively to a catheter, a needle may be used to introduce the cells into the defect.

A suspension is formed when an insoluble component mixes with a solvent. In this particular invention, a suspension is formed when the cells to be applied to the defect are mixed with the cell adhesion buffer. The suspension is then applied to the defect.

The term "cation dependent way" is intended to mean a binding between a cell adhesion molecule and its receptor (or ligand) which is dependent on divalent metal- ions such as Ca²⁺, Mg²⁺, and Mn²⁺.

The term "matrix" is intended to mean extracellular matrix proteins of the tissue such as collagens and proteoglycans. Matrix is secreted by the implanted cells (once they are bound at the defect site) and surrounds the cells. In the case of cartilage, the matrix consists mainly of collagens and proteo-glycans. In the case of bone, matrix is composed predominantly of collagens, proteo-glycans, phospho-proteins, and minerals.

The term "defect area" is intended to mean injured articular cartilage or bone which is surrounded by normal cartilage or bone. The term "defect cavity" \s intended to mean a "hole" in articular cartilage or bone. A defect cavity is created by excision of a chondral lesion during second arthroscopic operation (fig.2a) of articular cartilage or bone. A defect or defect cavity may occur in cartilage or bone when it is damaged (e.g. through disease or physical injury).

An endoscope is a device, which can be used to look at the surface of bone or cartilage tissue without the need for open surgery (i.e. through a portal in the skin of the subject). An arthroscope is a particular type of endoscope, which is used in the examination of joints.

An arthroscope according to the present invention is an instrument (a type of endoscope) which is inserted into a joint for visual examination. An arthroscope usually comprises a tube containing fibre optics, a lens and a light source, and allows joints to be examined without open surgery.

The term "scaffold' is intended to mean a temporary arrangement of proteins and/or synthetic material which may be implanted together with cells and provides a structure which they can grow around or inside. Scaffolds can provide both physical protection and structural form to the cells and support the adhesion of cells within the defect cavity.

The term viscosity is intended to mean internal friction of a moving fluid. A fluid with large viscosity resists motion because molecules in the fluid gives it a lot of internal friction and vice versa, a fluid with a low viscosity flows easily because of little friction when the molecules in the fluid is in motion. Viscosity is measured with a Viscometer. Viscosity is temperature dependent and typically decreases as the temperature rises. Water has a fairly low viscosity.

Viscosity can be measured in different units systems. The SI unit is N s/m², also known as the poiseuille (PI). An older unit, poise (dyne s/cm²) remains in common usage.10 poise equal N s/m² and 100 centipoise equal 1 poise. One Pa s or Pascal second can also be used.

The term "gel materiaf is intended to mean a solution of proteins (e.g., collagen) or other synthetic components in a more solid form than a solution. A gel material may also be implanted together with cells and may provide nourishment or physical protection to the cells.

The term "portaf is intended to mean a small operating procedure in which a minor hole is created in the skin in order to introduce an arthroscope as well as arthroscopic devices into articular joint.

The term "cartilage explanf is intended to mean a part of a mammalian articular cartilage, which has been explanted from a mammal by use of a suitable instrument. An explant may contain more than one kind of tissue, e.g., in the case of explantation of tissue from the knee, the explant may contain cartilage tissue as well as bone tissue. The explant can be cut into several pieces for the purpose of culturing the explant in a tissue culture flask. For some purposes it may be desirable to use a specific part of the explant. Normally, the part is collected from a mammal and used to produce one or more cell colony forming units (CFUs). By aid of certain factors in a growth medium, cells are able to migrate from the piece of mammalian tissue explant out into the growth medium. In the growth medium as well as in the explants, the cells start to proliferate and thereby form CFUs.

Colony Forming Unit (CFU)

The term "CFU" is intended to mean a colony of cells having the same origin, i.e., being derived from one cell such as, e.g. an cell which has migrated out from a piece of mammalian tissue explant and started divide to produce cell colonies outside the piece of mammalian tissue explant. The CFUs contain several layers of cells, the first one being adhered to the surface of the tissue culture flask in which the CFU is expanded and grown. The CFUs increase in size both vertically, by increasing the diameter of the CFU, and horizontally.

Cell adhesion is a property of in vivo or vitro cultured cells and influences cell morphology, mitosis and differentiation.

The sedimentation and adhesion of cultured cells to substrata (e.g., matrix) is an important consideration in designing methods for implantation of autologous cultured chondrocytes, chondroblasts, osteocytes and osteoblasts. Depending on the application of the cultured cells to a defect area, there are different requirements for optimal cell adhesion. Chondroblast/chondrocyte- and osteoblast/osteocyte-matrix interaction is mediated by a series of naturally-produced adhesion molecules, which includes: Integrins, cadherins, Ig family members, selectins, syndecans and CD44.

Binding of cell-surface receptors to matrix ligands at the defect surface appears to be fundamental in the initial adhesion of the implanted cells. Receptors on articular chondrocytes which mediate binding to these matrix molecules are for instance integrin-mediated, involving various integrin heterodimers such as α1β1, α3β1α, α5β1, α10β1 , αv1 β3, and αvβ5.

Articular chondrocytes have been shown to adhere in vitro to articular extra-cellular matrix proteins such as type II collagen, type VI collagen, laminin, fibronectin, gamma- carboxyglutamic acid protein, chondroadherin, vitronectin, osteopontin, and bone sialo- protein. In this context, cyclic RGD peptides are capable of inhibiting attachment of chondrocytes to various matrix molecules - an inhibition which is mediated through the integrins. Furthermore, the integrin-mediated adhesion to the mentioned matrix molecules has been shown to be dependent on cations such as Ca²⁺, Mg²⁺, and Mn²⁺, being present.

In order to obtain optimal binding between the graft cells (cultured chondrocytes, chondroblasts, osteocytes and osteoblasts) and the host tissue (articular cartilage and/or subchondral bone) during Arthroscopic ACI, several factors are considered in this invention: 1) Culture conditions of cells, 2) Harvesting method of cells, 3) Suspension of harvested cells into a "Cell Adhesion Buffer", 4) Preparation of damaged and degenerated articular cartilage or bone, 5) Injection of cells through a catheter or needle into the defect cavity, 6) Sedimentation of cells to a defect area by gravity forces and 7) Final adhesion of cells to the defect area (cartilage and bone).

The mechanism of cell adhesion to a defect area may be influenced by the culture conditions used prior to implantation. Culture conditions affect expression of integrins, cadherins, CD44 as well as other cell adhesion molecules.

The invention will now be described in detail with reference to the accompanying figures. Figure 1a illustrates the first arthroscopic operation - harvesting of a cartilage biopsy for use in cell culturing. Fig. 1b shows the front projection of the knee and arthroscopic portals.

Figure 2a shows the second arthroscopic operation - excision of the chondral lesion. Again, Figure 2b shows the front projection of the knee and arthroscopic portals. Figure 2c shows an expanded schematic view of the excision process, using an arthroscopic shaver and scalpel.

Figure 3a shows an embodiment of the invention, in which the knee is bent at 110-120° so that a cell suspension can be applied to a defect cavity in the tibia plateau or in the upper femoral condyles. Fig. 3b illustrates schematically the application of the cell suspension to the defect.

Figure 4 shows an alternative embodiment of the invention, wherein the cell suspension is injected into a defect cavity in the tibia plateau, and in which the knee is bent at ca. 90°.

Figure 5 shows an alternative embodiment of the invention, in which the knee is not bent, so that a cell suspension can be injected into a defect cavity in the patella or the femoral condyles.

Figure 6 is a schematic representation of the apparatus used in Example 1 , in which cultured porcine chondrocytes are implanted into a porcine cartilage defect using arthroscopy.

Figure 7 is a picture of a porcine femoral chondyle used in Example 2, showing chondrocyte sedimentation and adhesion to a full-thickness articular defect.

"First arthroscopic operation" - harvesting of cartilage biopsy for cell culturing (Figure 1a, 1b)

Arthroscopic Autologous Chondrocyte Implantation is a surgical method that involves fiberoptic camera technology (110) for visualizing and repairing of articular cartilage injuries with cultured chondroblast/chondrocytes or osteoblasts/osteocytes. The surgeon creates 3 small portals (116) with a diameter of about 5mm through the skin and muscle, into for instance the knee joint (comprising femur (114), fibula (128), patella (132) and tibia (126)), without a larger surgical incision (fig.1a, 1b). One portal (fig.1a) is created for the arthroscope (118) with magnifying lenses. Images from these lenses are sent to a television monitor (112), which the surgeon uses to monitor the procedure.

In the same portal, physiological salt water is pumped through the arthroscope using a pump (120) from a reservoir (130) into the joint, which inflates the knee joint. The pumping of salt water is necessary in order to 1 ) Distend the joint, which allow the surgeon to see better and 2) Flush out any cartilage and bone debris created by the arthroscopic procedure.

The other two portals (116) are placed on each sides of the knee (fig.1 b). Their functions are related to removing cartilage biopsies with arthroscopic devices (122).

Prior to preparation (excision) of damaged cartilage involving arthroscopic ACI (see below) the surgeon uses an arthroscope for harvesting of cartilage biopsies for cell isolation and culturing. Cartilage slices (100-300 mg) from e.g., a minor load-bearing area involving the upper medial femoral condyles of the knee are obtained with an arthroscopic scalpel (124).

The cartilage slices are stored and transported in sterile medium containing antibiotic. In a classified cell culture laboratory, the cartilage slices are further processed by the explant cell culturing method - for propagation of cartilage cells.

Cell culturing

We have found that cells (e.g. chondroblast/chondrocytes, osteoblast/osteocytes) that are isolated and grown with the explant culture method are superior in order to isolate sufficient numbers of migratory cells with the optimal adhesion properties for

Arthroscopic ACI, although other methods such as monolayer cultures and suspension cultures known to those skilled in the art are acceptable for the production of cells for use in the current invention.

With the explant culture method, chondroblast/chondrocytes and osteoblast/osteocytes are isolated by chemotaxi stimulation in the matrix. These cells are capable of migrating in the matrix towards the articular surface and further show adherence to various matrix molecules including articular cartilage and subchondral bone surface. The method is the same as that published in International Patent Application WO 02/061052 and is a production plant method of producing cell colony forming units in vitro from a mammalian tissue explant, comprising the steps of; a) growing a piece of the mammalian tissue explant in a growth medium to obtain cell colony forming units from immature cells from the piece of explant, and b) harvesting cells from one or more of the cell colony forming units for use in Autologous Cell Implantation/transplantation methods. The mammalian tissue explant in the above-mentioned method may be selected from the group consisting of cartilage such as elastic, fibro, hyalin or articular hyalin cartilage and bone such as, e.g., bone marrow. In one aspect, the present invention relates to a method for treating bone or cartilage defects as described herein, wherein the cells are obtained by: i. obtaining a tissue explant from a mammal, ii. culturing chondrocytes from said explant, iii. harvesting of said chondrocytes.

The cultured chondrocytes may be trypsinated before they are harvested, or between being harvested and implanted.

Alternatively, suitable cells for use in the present invention may be obtained by collagenase digestion of cartilage for isolation of chondroblast/chondrocytes in order to culture cells in a monolayer culture system or in a suspension culture system.

Flow cytometric analyses of human explant cultured chondrocytes have shown that these cells are highly CD44/CD13 double positive.

Due to the synthesis and deposition of extra-cellular matrix component around the cells during culturing, trypsination of the cultured cells is important in order to avoid steric blockade (masking) between the implanted cells and the defect surface after implantation.

Furthermore, it is critical to implant the harvested cells soon after trypsination in order to avoid subsequent deposition of matrix component on the cell surface as well as cell- cell interactions. If implantation of cells is not performed immediately after the first harvesting, a second trypsination can be introduced prior to implantation in order to secure that cells are free of extracellular matrix, which will otherwise hinder cell adhesion to the host tissue.

After trypsination of the cultured cells, the cells are re-suspended in a Cell Adhesion Buffer, which improves cell adhesion to the surface of the bone or cartilage.

"Second arthroscopic operation" - excision of chondral lesion. (Figure 2a, b, c)

In the case of cartilage defects, the chondral lesion has to be excised as far as the normal surrounding cartilage but not as far as the subchondral bone plate. In order to avoid generation of "zone of necrosis" and other damage in the normal surrounding cartilage, the damaged/degenerated cartilage has to be removed, which may be carried out with an arthroscopic device with a cutting blade such as a scalpel (124).

The excision of cartilage is performed perpendicular to the articular cartilage surface (210). The cutting blade (124) may be joined to the arthroscopic device (118). Salt water is pumped from a reservoir (130) using a pump (120) through the joint and out into a waste reservoir (136).

During arthroscopy of the wound knee joint (fig.2a, 2b, 2c), the surgeon may utilize a powered instrument (shaver (134)) to assist in the arthroscopic "cleaning" of articular cartilage. In this procedure, the surgeon removes damaged cartilage in the defect. All debris from this procedure is removed with a suction device connected to the shaver (fig.2c). Only necrotic cartilage (and bone) is removed with this device in order to avoid "zone of necrosis" (cell death in cartilage caused by apoptosis and necrosis) towards healthy cartilage in the defect area. "Zone of necrosis" hinders proper binding between graft cells and host tissue.

Injured cartilage (212) bordering healthy cartilage (214) is only excised with an arthroscopic scalpel (124) instead of "shaving" (see fig.2c for further details on how to prepare and excise damaged cartilage for arthroscopic ACI). In this context, a ¹/2-1 cm wide zone of injured cartilage is untouched before excision with the arthroscopic scalpel (fig.2c) takes place.

In short, the chondral lesion(s) in a particular joint is carefully excised with the arthroscopic scalpel as far as towards healthy surrounding cartilage but not as far as the subchondral bone plate (216, fig. 2c). Excision of cartilage is done in a tourniquet controlled bloodless field, while the patient is under general anesthesia.

All debris is cleared from the joint by inflow of salt-water and suction (larger cartilage pieces are removed with an arthroscopic device as shown in fig.1a) before arthroscopic implantation of cultured chondrocytes.

The cell suspension

Several studies have demonstrated that cell adhesion to cartilage matrix is Mg²⁺, Ca²⁺, as well as Mn²⁺ - dependent. Therefore, the cell suspension according to the present invention may contain ions selected from the group comprising Ca²⁺, Mn²⁺ or Mg²⁺ ions, or mixtures thereof. The cells are therefore re-suspended in a Cell Adhesion Buffer (cell suspension) comprising autologous or heterologous serum, culture medium and divalent metal-ions such as Ca²⁺, Mg²⁺, and Mn²⁺. Such ions may be present in the suspension in concentrations of 1μM - 10mM, such as e.g. 10μM - 10mM, 100μM - 10mM, 0.5mM - 10mM, 1mM - 5mM. The cell suspension may be made up of cells which are suspended in a medium selected from the group comprising; autologous serum, saline buffers, culture medium supplemented by divalent metal-ions. In an embodiment of the present invention, the cells are autologous. Arthroscopic implantation of cells takes place 3-5 weeks after the "first arthroscopic" operation (harvesting of biopsies). In short, the cell laboratory forward to the hospital/clinic, a syringe containing a suspension of cells (3,3 x 10⁶ cells/ml) in a "Cell Adhesion Buffer" containing; DMEM medium with 10% autologous serum, Fungizone, Gentamycin, Ascorbic acid 2-phosphate, supplemented by divalent metal ions reaching a final concentration for each salt as; 2mM MgCI₂, 2mM CaCI₂, and 0,1 mM MnCI₂.

Other factors which influences sedimentation and binding of implanted cells is related to the viscosity of the cell suspension as well as the surface tension. If the viscosity of the cell suspension is to low, fluid will flow out of the defect area and vice versa, if the viscosity is to high, sedimentation and binding of cells to the cartilage or bone will "delayed" or fail. It is important that the cell suspension according to the present invention has a viscosity which allows it to be injected via a canula or needle. In this invention, the cell suspension is composed of a 10-20% protein solution in a growth medium containing serum proteins and divalent metal ions, which do not form gel-like material, polymerize, or form other strong interactions with the cells prior to, during or after application. This solution contains only single cells and "free" molecules, which do not obstruct the binding of the cells to cartilage and bone in the defect cavity.

Depending on the local environment during implantation, a higher viscosity (relative to water) of the cell suspension (Cell Adhesion Buffer) can be obtained by adding reagents such as; NaCI, CaCI₂, MgCI₂, MnCI₂, poly-L-lysine, glycerol, ficoll (non ionic synthetic polymer of sucrose), hyaloronic acid or by increasing the concentration of serum proteins in the Cell Adhesion Buffer. A suitable viscosity range of the Cell Adhesion Buffer is between 0-10, such as between 0-3 mPa s at 25°C.

The implantation of single cells in a liquid suspension is superior compared to other ACI methods wherein for instance, cultured cells are implanted with fibrin glue, collagen gel or other gel-like materials. It is important that the cells are in suspension, as this allows them to settle into the defect under the influence of gravity. This means that no structure, scaffold, framework or other support matrix (e.g. a collagen matrix) is normally employed to support or protect the cells during implantation. If cells are implanted together with gel-like materials or other scaffolds (which is not the case in this invention), the binding between graft cells and host tissue might be hindered by the presence of "foreign" biological material. The outcome of cell implantation with gel materials or other scaffolds is poor or failure of integration of graft cells/matrix with host tissue. Likewise, the cell suspension, which is applied to the defect, does not undergo hardening, stiffening or solidification after being applied to a defect. In addition, the present invention does not require the use of binding agents such as adhesives, or the use of other binding means such as suturing of a membrane (periosteal or collagen l/lll membrane) to keep the cells within the defect.

Likewise, the bone or cartilage defect does not need to be treated with a binding agent before application of the cells. The cells themselves bring about adherence to the defect by adhesion molecules (e.g., integrins, cadhesins, CD44).

A (non-toxic) dye for visualization of the cell suspension during implantation can optionally be added to the Cell Adhesion Buffer. In order to improve visualization of the cell implantation procedure on the monitor during injection of cells, a non-toxic dye (e.g. food-dyes) such as; Phenol Red, Alamar Blue, Brown HT, Allura Red AC or Azorubin or other azo-dyes can be added to the cell suspension prior to implantation. These dyes should have no interference with the binding of the cells to cartilage and bone. Hence, in one embodiment of the present invention, the cell suspension contains at least one component, which imparts colour to the suspension (i.e. a dye or colouring agent). This helps the operator of the arthroscope to visualise the cell suspension as it is applied to the defect, and to direct its flow into the defect.

"Second arthroscopic operation" - implantation of cells. (Figure 3a, b, 4, 5)

Immobilisation of the defect

Prior to cell implantation, the patient with the particular defect (310) is immobilised on the operating table and positioned in such a manner that the plane of the defect

(measured according to the subchondral bone plate) is substantially horizontal to the operating table. This procedure is necessary to obtain proper sedimentation of cells to the defect area, and orients the defect (310) in such a manner that the cell suspension substantially does not flow out of the defect upon application of the cell suspension. In this context, the defect surface (measured according to the subchondral bone plate) and the operating table must be parallel to each other or at most at an angle of plus/minus 0-45°. This often means that the articular surface of the defect is substantially horizontal, i.e. with the surface parallel to the operating table/room, but can mean that the articular surface at the base of the defect is aligned for instance at most 45°, at most 30°, at most 15° to the horizontal. The correct alignment is accomplished by choosing the position of the defect that gives this orientation. For instance, if an articular cartilage defect on the lateral or medial condyle of the femur (114) has to be treated with Arthroscopic ACI, then the knee is flexed (about 130°) on the patient on the operating table (316) with femur (114) in a perpendicular position relative to the operating table.

In fig. 3a, 4, and 5, examples are described wherein the patient is bending the knee 110-120°, 90° and 0° accordingly, to position the cartilage defect (310) close to a horizontal level. This alignment is optimal. However, it is also feasible to carry out arthroscopic cell implantation with the defect surface (310) at an angle up to 45° (see fig.3a) relative to the operating table (316).

Cell implantation

The scope is placed close to the cartilage defect while the outlet of the catheter (312) is placed in the defect cavity (fig. 3a, 3b, 4 and 5). The flow of sterile salt-water into the joint is now turned off in order to create optimal conditions for cell sedimentation as well as cell adhesion to the various surfaces involving cartilage and subchondral bone plate.

The injection of cells through a portal into a defect cavity is mediated through a thin catheter or needle (312), which may be anchored to an arthroscope (118) or placed into the defect cavity (310) separately. With the former method, the orthopaedic surgeon is able to follow the whole procedure during cell implantation. With the catheter not anchored to the scope, the surgeon is able to position (with the scope) the catheter in even smaller joints, where the arthroscope is too large for penetration. Examples of such joints are the knee, elbow, ankle, wrist, hip, shoulder, jaw, spine, finger or toe joints, or any joint in the hand or foot. The defect is not necessarily completely filled with cells. In a typical example, a syringe loaded with a 1 ml cell suspension (concentration; 3,3x10⁶ cells/ml) in a Cell Adhesion Buffer is slowly emptied via the catheter into a 3,3 cm² defect cavity (fig.3b).

Without any disturbance of the cell suspension in the cavity, the arthroscope and catheter are now carefully removed from the joint. The arthroscope and catheter can also be removed later in the process, if desirable.

After injection of the cells into the defect cavity, the cells are allowed to settle

(sediment) into the cartilage or bone defect cavity under the influence of gravity. This sedimentation procedure is most critical in order to obtain a sufficient number of cells adhering to the surface. Therefore, all unnecessary disturbance of the defect, such as flow of saline into the joint during arthroscopy, has to be avoided at this point. While this sedimentation is occurring, it is important that the cartilage or bone defect is held substantially immobile.

Cell adherence

After implantation, the cells synthesize matrix proteins such as collagens and proteoglycans, which mediate the first binding (covalent as well as non-covalent) to the adjacent hyaline cartilage. This binding is highly critical for the successful outcome of the repair process. Final adhesion of cells to the subchondral bone and cartilage (fig.3b) takes place over a period of from 1 minute to 24 hours, such as e.g. from 5 minutes to 12 hours, from 15 minutes to 6 hours, from 30 minutes to 3 hours, from 45 minutes to 2 hours, from 50 minutes to 90 minutes such as e.g. 60 min. In this period, the defect is held substantially immobile, while adhesion of cells to subchondral bone and cartilage increases with time. After this period, the cells are resistant to detachment by gravitational forces and other handling. After this time period, the portals are closed.

The ideal number of cells per square centimetre in the defect after adherence to the defect is from 1x10⁵ to 1x10⁷ cells/cm², and the cells in the cell suspension are present in a concentration of 1x10⁵ to 1x10⁷ cells/ml, such as e.g. 1x10⁵ to 5x10⁶ cells/ml, 5x10⁵ to 5x10⁶ cells/ml, 1x10⁶ to 5x10⁶ cells/ml.

It has been shown by others (see Melissa S Kurtis, Tannin A Schmidt, William D Bugbee, Richard F Loeser, Robert L. Sah. Integrin-mediated adhesion of human articular chondrocytes to cartilage. Arthritis and Rheumatism. Vol.48, no.1. 2003. pp.110-118), that pre-treatment of chondrocytes with monoclonal antibodies against certain integrins, blocks adhesion of human articular chondrocytes to cartilage. These results strongly indicate that integrins are important for the first contact between the chondrocytes and the matrix. In this context, it has been shown that the binding of integrins to certain matrix proteins are dependent on cations such as Ca²⁺, Mg²⁺, and Mn²⁺and further, that binding between integrins and ligands (matrix proteins) are blocked with cyclic synthetic peptides containing a RGD recognition motif. In order to obtain optimal cell adhesion during arthroscopic injection of chondrocytes, the chondrocytes are re-suspended in the Cell Adhesion Buffer containing e.g. 2 mM MgCI₂, 0.1 mM MnCI₂, 2 mM CaCI₂, in DMEM/F12 medium containing 10 % autologous serum.

Subsequent immobilisation

After arthroscopic implantation of the cultured cells, the surgically treated defect should be immobilized for a short period of about 1-48 hours after the cells have been allowed to settle, such as e.g. 2-36 hours, 5-24 hours or 10-24 hours and further protected from strong motion and loading for a similar amount of time in order for the cells to secrete matrix into the defect area, which will mediate binding to the adjacent bone or cartilage as well as protecting the implanted cells from mechanical disturbance. This further immobilisation usually means that the patient is confined to a bed and active movement of the articular joint without weight bearing is only initiated after this period. After cell binding and (limited) matrix synthesis in the defect cavity, immobilization of the defect can have any angle. It is only during application of the cell suspension that immobilization of the joint (at a particular angle) has to take place, in order to secure proper sedimentation of the cells in the defect area.

With the arthroscopic ACI treatment method, it is feasible to inject more cells into the cartilage defect area in further operations. In this way, more than one "layer" of cells can be established in the defect area. These "layers" of cells can either be introduced under the same operative procedure by repeating the steps of cell sedimentation and adhesion (above) or they can be introduced during multiple arthroscopic ACI treatments within days, months or years.

As mentioned earlier, conventional ACI uses a periosteal flap or patch to cover the cells and keep them in the defect. The method according to the present invention, however, allows treatment or repair of cartilage or bone defects without the use of a periosteal flap/patch, as the cells settle into the defect under the influence of gravity and adhere to the cartilage or bone while the defect is immobilised.

Suitable subjects for treatment under the method according to the present invention are mammals, such as human, horse, camel, dog or cat.

There follows a series of examples which illustrate preferred embodiments of the present invention. The examples are only meant as guidance, and one skilled in the art will know how to modify them to obtain the desired result.

Examples

Example 1

In vitro implantation of cultured porcine chondrocytes into a porcine cartilage defect using arthroscopy

Creation of a full-thickness articular defect in the femoral condyle of the porcine hind limb knee

A left hind limb (610) from a healthy one-year old pig was obtained from a local slaughterhouse (Steff Houlberg/Danisco, Ringsted, Denmark). The limb was transported to the laboratory in a cooling box (with ice) prior to the experiment. In the laboratory, the limb was anchored to a rack (612) with various catchers (614) according to fig.6. In short, the limb was placed upside down in the rack with the femoral condyles pointing upwards. The special orientation of the limb was arranged in order to orientate part of the femur condyles parallel with the operating table.

Two small incisions (portals, 116) were made anterolateral and anteromedial in the porcine knee. One incision was used for the catheter (312) and the scope (118) with a "two-connect sheath system" (Linvatec's pressure sensing pump systems, Quicklatch™) according to fig.6. The second incision was used for the outflow tubing of the fluid. A third incision was made on the lateral side of the porcine knee, distal to the other two portals. The third portal (116) was used for arthroscopic devices for creating a full-thickness articular defect into the femoral condyle.

The pump (120) was turned on in order to flow saline from a reservoir (130) into the knee joint and out into a waste container (136).

A one cm² full-thickness cartilage defect was created in both the lateral and medial femoral condyles using arthroscopic devices and a scope (118) (Quicklatch™) connected with a camera head (110) (EnVision ™). In these defects, cartilage was excised to the subchondral bone with an arthroscopic scalpel (in example 1 , an arthroscopic shaver was not used). The full-thickness cartilage defects were made perpendicular to the cartilage surface and all cartilage was removed towards the subchondral zone as shown in fig.2c.

Arthroscopic harvesting of cartilage biopsy for cell culturing During creation of the full-thickness defect into the articular surface, smaller cartilage pieces were flushed out with the salt-water flow system while larger pieces were taken out with an arthroscopic device and used for culturing cartilage cells according to the explant culturing method.

Culturing of porcine chondrocytes with the "explant culturing method"

Cartilage biopsies taken out with the arthroscopic device were dissected with a scalpel under sterile conditions in a cell culture laboratory. Each cartilage specimen was further divided into smaller pieces by cutting perpendicular to the articular surface. Explants, measuring about 5 x 5 x 3-5 mm, were used in the following experiments for propagation of chondrocytes. Explants were cultured in 75 cm² cell culture flasks (InVitrogen) (5 explants in each flask). Dubelcco's Modified Eagle's Medium DMEM/NUT.MIX.F-12 medium with Glutamax-1 (InVitrogene) with 20 % Fetal Calf Serum (FCS) (Life Technologies) Gentamycin (49 ug/ml), Fungizone (1,2 ug/ml), Ascorbic acid 2-phosphate (87 ug/ml), were added (30 ml pr culture flask). Reagents were obtained from Sigma Aldrich.

Fresh medium was added every 3rd day to all explants in order to stimulate chondrocytes in the explants for migration (chemotaxi) into the culture medium following chondrocyte proliferation and formation of Colony Forming Units (CFU). After 3-4 weeks of explant culturing, about 2 x10⁶ porcine chondrocytes were propagated from each culture flask. The chondrocytes were harvested using 0.25% Trypsin/EDTA solution and re-suspended in an "Cell Adhesion Buffer" containing 10% Fetal Calf Serum in DMEM/F12 medium supplemented with 2 mM CaCI₂, 2 mM MgCI₂ and 0.1 mM MnCI₂.

Arthroscopic implantation of chondrocytes into a full-thickness articular defect Prior to arthroscopic chondrocyte implantation using the porcine hind limb knee, the scope (118) was placed immediately in the vicinity of the cartilage defect (310) with the outlet of the catheter (312) positioned in the middle of the defect cavity as shown in fig.3b and fig.6.

The flow of saline into the knee joint was turned off in order to create the proper conditions for implantation of the cultured chondrocytes. This means that fluid does not disturb the final implantation procedure, which involves chondrocyte sedimentation and adhesion to the subchondral bone as well as to the four articular surfaces making up the cartilage defect (fig.3b).

The volume of chondrocyte suspension required for repair of a defect of 1 cm² was estimated as follows: The thickness of the cartilage was estimated to be about 0,3 cm, x 1cm x 1cm = 0.3 ml. The optimal concentration of chondrocytes adhering to a 1cm² cartilage defect was in example 2 (see below) estimated to be 1x10⁶ chondrocytes/cm². This means that a total concentration of 3,3x10⁶ chondrocytes/ml is needed in the final chondrocyte suspension for implantation.

The suspension (0.3 ml) of cultured chondrocytes in the syringe (314) was slowly emptied into the defect cavity (310) through the catheter (312) according to fig.3b. Due to gravitational force, the chondrocytes in the defect cavity show sedimentation after 5-10 min and optimal chondrocyte adhesion to the subchondral bone surface and cartilage surfaces was obtained after 30-60min.

After final chondrocyte adhesion, the scope (318) and the catheter (312) were slowly removed from the joint and the three portals (116) were closed.

Example 2 In vitro study of chondrocyte sedimentation and adhesion to a full-thickness articular defect, in the porcine femoral condyle

Porcine left hind limbs from "healthy" one-year-old pigs were obtained from a local slaughter-house (Steff Houlberg/Danisco, Ringsted, Denmark). In the laboratory, the femoral condyles (718) were "amputated" from the exposed knees with a saw and a scalpel. An amputated condyle is shown in fig.7 (718). The femoral condyles obtained by this procedure were used in the following experiment, 3 hours after the pigs were slaughtered.

Porcine chondrocytes were cultured according to the explant cell culturing method described in example 1. In short, 10x10⁶ porcine chondrocytes were harvested from a 75 cm² culture flask with 0,25%trypsin/EDTA and re-suspended in a Cell Adhesion Buffer" (1ml) as described in example 1.

The chondrocyte suspension containing 10x10⁶ chondrocytes/ml was carefully loaded into a 1 ml syringe with a 16 Gauge needle. The syringe was placed into a 37°C incubator supported with 5% CO₂ prior to in vitro implantation.

Prior to in vitro chondrocyte implantation, a 1 cm² full-thickness cartilage defect (710), measuring about 0,75cm x 1 ,35cm, was created in the middle of the medial and lateral femoral condyles using a sterile scalpel (fig.7).

The full-thickness cartilage defects (710) were made perpendicular to the cartilage surface and all cartilage was removed towards the subchondral bone plate (712, fig.7).

The femoral condyles were washed in DMEM/F12 medium and transferred into sterile petri dishes (714) containing DMEM/F12 medium with 10% porcine serum (716). The petri dishes (with lid on) were then transferred into a 37°C incubator supported with 5% CO₂. The femoral condyles with the defect area pointing upwards and parallel to the table were equilibrated in the incubator for ¹ hour. These conditions were selected in order to imitate physiological conditions.

After equilibration, the medium in the petri dish was removed and 0,3 ml (0,3 ml x 10x10⁶ chondrocytes/ml = 3 x 10⁶ chondrocytes/cm²) of re-suspended chondrocytes were loaded into the defect, in the incubator.

The petri dish was incubated for 1 hour at 37°C in order to obtain sedimentation and adherence of the chondrocytes into the defect cavity. After 1 hour of incubation, the femoral condyles loaded with explant-cultured chondrocytes were washed several times in the petri dish with 37°C Cell Adhesion Buffer in order to remove "non-binding" chondrocytes in the defect cavity.

After this procedure, the femoral condyles were further washed in a second system. In this system, each femoral condyle was transferred into separate 50 ml Falcon tubes filled with DMEM/F12 medium containing 10% porcine serum. The filled tubes were anchored to a horizontal shaker in the CO₂ incubator and set to "level 1" for 60 minutes.

After "final washing" at 37°C, the supernatant was removed and the number of chondrocytes adhering to the defect was counted in the microscope by adding 0.3 ml of 0.25% trypsin/EDTA for 5 min to the defect cavity.

Results showed that an average number of 1x10⁶ chondrocytes pr cm² full-thickness defect was obtained after harvesting chondrocytes from several 1cm² defect cavities, in order to release the cells.

Claims

1. An endoscopic method for treating cartilage or bone defects in a mammal, said method comprising the steps of: i) identifying the position of the defect, ii) applying a cell suspension comprising cells selected from the group consisting of chondrocytes, chondroblasts, osteocytes and osteoblasts and combinations thereof into the cartilage or bone defect, the defect being positioned in such a manner that the cell suspension substantially does not flow out of the defect upon application.

2. A method according to claim 1 , wherein the cells in the cell suspension are allowed to settle into the cartilage or bone defect under the influence of gravity after application.

3. A method according to any of the preceding claims, wherein after settling into the defect, the cells are allowed to adhere to the surface of the cartilage or bone defect by adhesion molecules.

4. A method according to claims 2 or 3, wherein the cartilage or bone defect is held substantially immobile while the cells settle into and adhere to the cartilage or bone defect.

5. A method according to claim 4 wherein the cartilage or bone defect is held substantially immobile over a period of from 1 minute to 24 hours, such as e.g. from 5 minutes to 12 hours, from 15 minutes to 6 hours, from 30 minutes to 3 hours, from 45 minutes to 2 hours, from 50 minutes to 90 minutes, such as e.g. 60 minutes.

6. A method according to any of the preceding claims, wherein the cartilage or bone defect is held substantially immobile for a further 1-48 hours after the chondrocytes have been allowed to settle, such as e.g. 2-36 hours, 5-24 hours or 10-24 hours to allow further adhesion and/or secretion of matrix into the defect.

7. A method according to any of the preceding claims, wherein the cell suspension is applied to the defect more than once, so as to build up more than one layer of cells in the defect.

8. A method according to any of the preceding claims, wherein the defect is positioned such that the articular surface of the defect is aligned at most 30°, such as at most 15° to the horizontal.

9. A method according to any of the preceding claims, wherein an arthroscopic device is used to identify the position of the defect.

10. A method according to any of the preceding claims in which a periosteal flap/patch is not used.

11. A method according to any of the preceding claims in which a framework, scaffold or other support matrix is not used to support the cells.

12. A method according to any of the preceding claims, wherein the number of cells per square centimetre in the defect after adherence to the defect is from 1x10⁵ to 1x10⁷ cells/cm².

13. A method according to any of the preceding claims, in which the cells in the cell suspension are autologous.

14. A method according to any of the preceding claims, wherein the cells in the cell suspension are present in a concentration of from 1x10⁵ to 1x10⁷ cells/ml, such as e.g. from 1x10⁵ to 5x10⁶ cells/ml, from 5x10⁵ to 5x10⁶ cells/ml, from 1x10⁶ to 5x10⁶ cells/ml.

15. A method according to any of the preceding claims, wherein the cell suspension is made up of cells which are suspended in a medium selected from the group comprising; autologous serum, saline buffers, culture medium.

16. A method according to claim 15, wherein autologous serum is re-suspended in Dulbecco's Modified Eagle's Medium (DMEM).

17. A method according to any of the preceding claims, wherein the cell suspension contains ions selected from the group comprising Ca²⁺, Mn²⁺ or Mg²⁺ ions, or mixtures thereof.

18. A method according to claim 17, wherein the cell suspension contains Ca²⁺ ions in a concentration of from about 1 μM to about 10 mM, such as e.g. from about 10 μM to about 10 mM, from about 100 μM to about 10 mM, from about 0.5 mM to about 10 mM, from about 1mM to about 10 mM or from about 1mM to about 5 mM.

19. A method according to claim 17, wherein the cell suspension contains Mn²⁺ ions in a concentration of from about 1 μM to about 10 mM, such as e.g. from about 10 μM to about 10mM, from about 100 μM to about 10 mM, from about 0.5 mM to about 10 mM, from about 1 mM to about 10 mM or from about 1mM to about 5mM.

20. A method according to claim 17, wherein the cell suspension contains Mg²⁺ ions in a concentration of from about 1 μM to about 10 mM, such as e.g. from about 10 μM to about 10 mM, from about 100 μM to about 10 mM, from about 0.5 mM to about 10 mM, from about 1 mM to about 10 mM or from about 1 mM to about 5 mM.

21. A method according to any of the preceding claims, wherein the cell suspension contains a colouring agent.

22. A method according to any of the preceding claims, in which the cell suspension contains additives which increase viscosity, such as e.g. NaCI, CaCI₂, MgCI₂, MnCI₂, poly-L-lysine, glycerol, ficoll, hyaloronic acid or serum proteins.

23. A method according to any of the preceding claims, in which the cell suspension has a viscosity of between 0-10, such as between 0-3 mPa s at 25°C.

24. A method according to any of the preceding claims, wherein the cell suspension is applied using a catheter or a needle.

25. A method according to claim 24, wherein an arthroscopic device is used to position the catheter or needle.

26. A method according to claim 25, wherein the catheter or needle is attached to the arthroscopic device.

27. A method according to any of the preceding claims, wherein damaged or degenerated cartilage around a cartilage or bone defect is removed by a cutting blade, such as a scalpel.

28. A method according to claim 27, wherein the cutting blade is attached to an arthroscopic device.

29. A method according to any of the preceding claims, wherein the cartilage or bone defect is located in a joint of the subject

30. A method according to claim 29, wherein the joint is selected from the group comprising the knee, elbow, ankle, wrist, hip, shoulder, jaw, spine, finger or toe joints, or any joint in the hand or foot.

31. A method according to any of the preceding claims, wherein the cells are obtained by: obtaining a tissue explant from the subject, culturing chondrocytes from said explant, harvesting said chondrocytes.

32. A method according to claim 31 , wherein the cultured chondrocytes are trypsinated before they are harvested, or between being harvested and implanted.

33. A method according to any of claims 1-30, wherein cartilage is digested with collagenase for isolation of chondroblasts/chondrocytes, which are cultured in a monolayer culture system or in a suspension culture system.

34. A method according to any of claims 31-33, wherein the subject is selected from mammals, such as human, horse, camel, dog and cat.