WO2005023324A1

WO2005023324A1 - Meniscal superfacial zone cells for articular cartilage repair

Info

Publication number: WO2005023324A1
Application number: PCT/GB2004/003810
Authority: WO
Inventors: Samuel James Boyer; Rebecca Turner; James William Huckle; Catherine Booth
Original assignee: Smith & Nephew Plc
Priority date: 2003-09-04
Filing date: 2004-09-03
Publication date: 2005-03-17
Also published as: GB0320691D0

Abstract

The present invention relates to isolated joint lining cells, especially meniscal superficial zone cells useful for the repair of damaged cartilage or other tissues within the joint, for example meniscus, tendon and ligament. In particular the present invention relates to methods for isolating meniscal surface zone cells, production of implants for articular cartilage repair, use of the cells for promotion of articular cartilage generation and methods for repairing defects in articular cartilage.

Description

MENISCAL SUPERFICIAL ZONE CELLS FOR ARTICULAR CARTILAGE REPAIR

It has long been recognised that injured cartilage does not readily heal. Partial-thickness cartilage defects trigger a minimal and ineffectual repair response whilst full-thickness wounds, (which penetrate the underlying bone), induce only a mechanically inadequate fibrocartilagenous scar tissue that degenerates over time. No fully effective clinical treatment is available. Popular current approaches for the treatment of articular cartilage defects include high tibial osteotomy (primarily performed in Europe) and muscle release to alter joint biomechanics and loading; lavage and debridement to remove osteophytes and fibrillated areas of cartilage and _^perforation and penetration of the subchondral bone to induce bleeding and clot formation. It has been established however that none of these techniques leads to successful regeneration of the tissue that duplicate the structure, composition, mechanical properties or durability of articular cartilage.

The most common technique is that of subchondral drilling which results in the formation of a fibrin clot and a fibrous tissue. In about 75% of patients there is a satisfactory result, but two years after the operation, only 12% of patients remain free of symptoms. Limitations of this approach are the difficulty in predicting the quality and duration of the clinical outcome and, in the long term, this procedure often leaves the patient in a worse condition. On the other hand, subchondral drilling and debridement are generally successful in alleviating symptoms and in postponing the requirement for total knee replacement by between one and three years. Other treatments include carbon fibre rods. Here the cartilage lesions are cleaned and excised down to the subchondral bone and the carbon fibre rods are inserted into holes made therein. Repair begins with the deposition of collagen around the top of the rods. These islands of growth soon enlarge to completely fill the defect, the rods providing anchorage for the repair tissue in the defect. A significant problem associated with this product is the production of carbon fibre wear debris found in the synovial cavity, which may give rise to problems of abrasion.

Although recent studies have highlighted the promise of tissue engineering or cell transplantation for cartilage repair, the ideal cell for tissue engineering of articular cartilage has yet to be identified. Such cell-based approaches, (e.g. Autologous Chondrocyte Transplantation), show potential but are constrained by an inadequate supply of differentiated human cartilage cells. A clinically and commercially viable, supply of human cartilage-forming cells would therefore constitute a major advance in the field of tissue engineering. The required cells would need to be (i) derived from a readily available and ethically acceptable human tissue source, (ii) highly proliferative, (iii) able to differentiate readily and consistently under appropriate culture conditions and (iv) immunologically tolerated by the recipient. Such cells might also be used in novel therapies for the repair of other connective tissues, e.g. meniscus.

Difficulties arise in the use of chondrocytes in tissue engineering processes due to the limited number of cell divisions a mature chondrocyte will undergo in vitro prior to the onset of senescence. The proliferative potential is dependent on species and inversely proportional to the age of the donor [Evans CH & Georgescu HI. Observations on the senescence of cells derived from articular cartilage. Mech Ageing Dev. 1983; 179-91]. The importance of this becomes apparent considering the limited number of chondrocytes that can be harvested from a patient and the large numbers of cells required for cell transplantation procedures and seeding tissue engineering scaffolds.

The limited growth potential of chondrocytes in culture is further complicated by the rapid loss of phenotype, known as dedifferentiation, of chondrocytes in monolayer culture [Benja PD & Shaffer JD. Dedifferentiated chondrocytes re- express the differentiated collagen phenotype when cultured in agarose gels. Cell. 1982; 30 215-24]. Dedifferentiation is characterised by a loss of the normal spherical cell morphology and concomitant decrease in collagen II and aggrecan synthesis to a fibroblastic-like morphology with up-regulation of collagen I, III and versican. Possible solutions to enable the rapid expansion of chondrocytes whilst retaining their phenotype have been suggested. Such studies usually involve the use of growth factors (e.g. bFGF, TGF-β) [Jakob M, et al. Specific growth factors during the expansion and redifferentiation of adult human articular chondrocytes enhance chondrogenesis and cartilaginous tissue formation in vitro. J Cell Biochem. 2001 ; Vol. 81 368-77] or the reversal of dedifferentiation, known as redifferentiation, by transfer into a culture format that supports a spherical cell morphology (e.g. pellet culture or alginate) [Bonaventure J, et al. Re-expression of cartilage-specific genes by dedifferentiated human articular chondrocytes cultured in alginate beads. Exp Cell Res. 1994; Vol. 212 97-104]. However the redifferentiation process is rarely complete and occurs to a lesser extent the longer the cell is maintained in monolayer culture.

Therefore, the identification of a cell with extensive growth potential and which retains its chondrogenic ability after extensive growth in culture would be a major step forward in addressing these issues. Mesenchymal stem cells (MSCs) from the bone marrow stroma have a high capacity for self-renewal and considered to be immunologically tolerable and therefore thought to be of great value for cartilage repair and other tissue engineering applications. The chondrogenic potential of MSCs is well characterised and the effect of various growth factors on the proliferation and differentiation of MSCs have also been studied. However, MSCs have been shown to generate repair tissues with varying degrees of success in in vivo cartilage repair studies [Wakitani et al. Mesenchymal cell-based repair of large, full-thickness defects of articular cartilage. J. Bone. Joint. Surg. 1994; Vol. 76 579-591].

Our previous studies which explored the biology of cartilage wound repair, identified a population of 'cartilage stem cells' in the superficial area of immature bovine articular cartilage [Patent WO 02/10348]. These progenitor cells appear to contribute to cartilage growth by supplying new chondrocytes to the developing mid zone. The superficial zone chondroprogenitor cells can be isolated due to their increased affinity for fibronectin and can be expanded ~ 10¹⁸ fold in monolayer culture before the onset of senescence, (> 35 million fold without loss of demonstrable chondrogenic phenotype) [Boyer, S et al Paper 102, Transactions ORS Vol. 28, New Orleans, Louisiana, 2003]. Although the cells of the superficial zone of the articular cartilage have many of the required properties of a cell for tissue engineering purposes (e.g. a high proliferative capacity in vitro and an ability to differentiate into mature cartilage) difficulties arise in sourcing suitable, healthy donor articular cartilage for the isolation of the cells. Additionally, cartilage is not an ideal donor tissue as its removal creates another defect that may need to be treated.

There are a number of other tissues in the joint area potentially including but not limited to fibrocartilage, costal cartilage, fat pad, pannus, ligament, tendon, synovium and meniscus.

The menisci are very important semi-lunar wedge shaped fibrocartilagenous structures situated between the tibia and femur. The menisci perform several functions in the knee including load transmission from the femur to the tibia, stabilization in the anterior-posterior position during flexion, and joint lubrication. Histologically, the meniscus is a fibrocartilagenous tissue composed primarily of an interlacing network of collagen fibres interposed with two distinct cell populations - cells found in the superficial layers and the cells deeper in the tissue. The cells deep in the bulk of the tissue are rounded, found in discrete lacunae and bear close resemblance to the chondrocytes of articular cartilage. However, these rounded cells in the deeper areas of the meniscus produce a predominantly Type I collagen rich extracellular matrix whereas those of the articular cartilage produce a predominantly Type II collagen rich extracellular matrix. Therefore the cells are phenotypically much different from articular cartilage cells. The superficial layers of the meniscus contain flattened and oval cells that lie perpendicular to the surface of the tissue. Huckle et al [Novartis Found Symp 2003; 249 103-12; discussion 112-7, 170- 4, 239-41] have attempted to use meniscal fibrochondrocytes seeded onto fibrous scaffolds in an attempt to generate tissue engineered hyaline-like cartilage for articular cartilage repair. However, it was necessary to apply dynamic hydrostatic pressure (30 minutes/day, 1 Hz frequency, 50 bar overpressure maximum) and the tissue generated was only vaguely hyalinelike, more closely resembling a fibrocartilage.

Surprisingly, we have found that a population of cells from the superficial area of joint lining tissues, for example meniscus, can be induced to form a hyalinelike articular cartilage matrix and so may be beneficial for articular cartilage repair. Such tissues are more readily available. For example healthy meniscal tissue from young donors is routinely removed during meniscectomies for the repair of bucket-handle tears. Thus, tissues such as meniscus would constitute an ideal allogeneic source of joint lining cells for tissue engineering.

In accordance with one aspect of the present invention there is provided isolated meniscal superficial zone cells useful for repairing articular cartilage.

According to another aspect of the invention there is provided isolated meniscal superficial zone cells useful for the promotion of articular cartilage generation.

The meniscal superficial zone cells may express CD105, Msx-1 , Stro-1 , BST- 1 , Fibronectin-EDA, α-smooth muscle actin or superficial zone protein. The superficial area is characterised as being preferably less than 500μm but ideally less than 150μm from the meniscal joint lining surface.

In accordance with another aspect of the present invention there is further provided a pharmaceutical composition comprising meniscal superficial zone cells. The pharmaceutical composition aptly comprises saline, buffered solutions or hyaluronic acid.

According to another aspect of the present invention there is provided a medicament, for the treatment of clinical conditions featuring or characterised by articular cartilage deficiency or injury, comprising meniscal superficial zone cells.

Accordingly, in another aspect of the present invention, there is provided the use of meniscal superficial zone cells in the manufacture of a medicament for the treatment of clinical conditions that may be alleviated by the promotion of articular cartilage generation.

There is further provided a method for producing a medicament containing meniscal superficial zone cells for the prevention of and/or treatment of cartilage defects.

In accordance with another aspect of the present invention there is further provided a medical device comprising meniscal superficial zone cells.

The device aptly comprises cells and a support medium. Suitable cells include meniscal superficial zone cells as hereinbefore mentioned. The support medium may be a scaffold, Synthetic (resorbable or non-resorbable) or natural (for example collagen, fibrin or hyaluronic acid) three dimensional scaffolds in a fibrous, gel, foam, particulate or sponge format. Alternatively a hydrogel, alginate or pellet culture could be used. Scaffolds could comprise polyglycolic acid (PGA), polylactic acid (PLA), co-polymers thereof or polyethylene terepthalate (PET) non-woven felt. The cells may remain on the surface of the support medium, but preferably substantially infiltrate said medium. The devices may be cultured for a period of time with or without mechanical loading or with intermittent or periodic mechanical loading. During any culture period the cells may remain in a static state or deposit growth factors and extracellular matrix.

In accordance with another aspect of the present invention there is further provided a method for preventing and/or treating cartilage defects comprising the application of meniscal superficial zone cells to articular cartilage defects.

Articular cartilage defects include focal defects, defects caused by osteo- or rheumatoid arthritis or defects caused by trauma or surgery. The cells may be administered in an undifferentiated state or be pre- differentiated prior to administration using suitable factors or culture conditions. The cells are preferably administered in a delivery vehicle. Suitable delivery vehicles include saline, buffered solutions or hyaluronic acid. Apt administration methods include direct application via open surgery or intra-articular injection, although the cells are preferably administered arthroscopically.

In accordance with another aspect of the present invention there is further provided a method of preventing and/or treating cartilage defects comprising the application of a device comprising meniscal superficial zone cells to articular cartilage defects

Suitable administration methods include direct application via open surgery or arthroscopic administration.

In accordance with another aspect of the present invention there is further provided a method for the promotion of articular cartilage generation using meniscal superficial zone cells.

In accordance with another aspect of the present invention there is further provided a method for isolating meniscal superficial zone cells.

Cells may be isolated using centrifugal techniques, for example ficol gradients or counter current elutriation; antibody binding, for example FACS or MACS); selective culture media or on the basis of the physical properties of the cell, for example size or granularity. Preferably the isolation method will be via differential adhesion to a particular substrate. Ideally this substrate is fibronectin. For an example of a suitable isolation method see Boyer et al (2003), Chondrogenic ability of articular cartilage progenitor cells during extensive sub-culture, Trans. ORS. 49, 102.

The invention will now be illustrated with reference to the accompanying examples and drawings. Example 1. Isolation of cells

Bovine plasma fibronectin (Sigma) was used at 10μgml^"1 in Dulbecco's phosphate buffered saline (PBS) with 1 mM MgCI₂ and 1 mM CaCI₂ (Sigma). 7.8mls of this solution was then added to 75cm² flasks and incubated overnight at 4°C for coating. The fibronectin solution was then aspirated and the flasks were blocked with 1% bovine serum albumin (BSA) prior to addition of cells.

Cells were isolated from the superficial area of the 2-3 week old bovine meniscus by fine dissection. The portions of tissue were placed directly into Dulbecco's Modified Eagle Medium (DMEM) containing 5% foetal calf serum (FCS) and 0.1% pronase (Merck, 4x10⁶ units/g) and incubated at 37°C for 3 hours. Tissue was then washed once with PBS and incubated in DMEM containing 5% FCS and 0.04% collagenase (Worthington, 237U/mg) and incubated overnight at 37°C with gentle shaking.

Tissue digests were strained through a 70μm cell strainer (Falcon) to remove debris. The resultant filtrate was centrifuged at 1000 rpm for 10 minutes to pellet cells and then resuspended in 10ml of serum free DMEM followed by centrifuging again at 1000 rpm for 5 minutes. The cell pellet was resuspended in 10ml serum free DMEM, cell number counted using a neubeuer haemocytometer and resuspended to a final concentration of 4000 cells/ml.

After isolation, 7.8ml of a suspension of 4000 cells/ml superficial area cells in serum free DMEM was seeded into a coated flask and incubated at 37°C for 20 minutes. After 20 minutes, the media was gently swirled and discarded. Fresh DMEM containing 10% FCS was added to each well. The flasks were incubated at 37°C in a 5% CO₂ atmosphere. When cells in the flasks were seen to be approaching confluency, media was removed and the monolayer washed with PBS. Cells were passaged by the addition of 1.5 ml trypsin-EDTA to each flask for 10 minutes. DMEM containing 10% FCS was then added and the media aspirated and pooled. The cell suspension was centrifuged at lOOOrpm for 5 minutes and the resultant pellet resuspended in 5mls DMEM/10% FCS. An aliquot of cells was removed for pellet culture and the remaining cells were transferred to a 25cm² culture flask (P1). Subsequent growth was maintained by continual passage at a ratio of 1 :3. Passaging occurred prior to the cells reaching full confluence to maintain the cells in the log phase of growth.

Cultures of normal fibrochondrocytes isolated from the full thickness of the bovine meniscus were also set up to serve as controls for the pellet culture experiments.

Example 2. Formation of articular cartilage from meniscal superficial area cells

Meniscal superficial area cells were induced to form cartilage using pellet culture. Cells were isolated and expanded as described in example 1. Aliquots of 200,000 cells were suspended in chondrogenesis media (DMEM supplemented with penicillin/streptomycin; ITS premix; ascorbate 2- phosphate, 100μM; dexamethasone, 10^"7M; and TGFβ-1 , 10ng/ml) and pelleted by centrifugation in polypropylene Falcon tubes. Pellets were incubated with the lids of the tubes loosened at 37°C in a 5% CO₂ atmosphere for 14 days with media changes carried out every 2-3 days. After culture, pellets were fixed overnight in 10% neutral buffered formalin and embedded in paraffin wax. Sections of 5μm thickness were cut and stained with Safranin O and haematoxylin and immunostained for the presence of collagen II.

For collagen II immunostaining, sections were dewaxed and then washed in PBS. Antigen retrieval was carried out using Pepsin and sections were blocked with 2.5% normal rabbit serum for 20 minutes at room temperature prior to incubation with 2 μg/ml mouse anti-collagen II antibody overnight at 4°C. Sections were washed with PBS and incubated with a 1 :100 dilution of biotinylated rabbit anti-mouse secondary antibody in PBS for 1 hour at room temperature. Finally, sections were washed again with PBS and incubated with a 1 :100 dilution of Streptavidin-FITC for 1 hour at room temperature, then mounted and coverslipped. Sections were viewed using fluorescence microscopy and photographed.

Bovine meniscal superficial area cells with a high affinity for fibronectin were expanded in culture and subsequently grown in pelleted micromasses (Figure 1 ). The cells synthesised a hyaline-like cartilage matrix that stained strongly with Safranin O, indicating the presence of sulphated proteoglycans (Figure 2). The periphery of the pellets stained weakly with Safranin O. In addition, flattened cells were seen to be present on the surface of the pellet. The cell pellets were rich in collagen II (Figure 3). Pellet cultures of normal fibrochondrocytes isolated from the full thickness of the bovine meniscus appeared to be smaller in volume (Figure 4) and histologically have less matrix than in superficial area pellets (Figure 5). Furthermore, the matrix contained negligable collagen II (Figure 6).

Hence, whilst the normal meniscal fibrochondrocytes produced a pellet that contained no collagen II, the meniscal superficial area cells produced a hyaline-like, sulphated- proteoglycan rich matrix that stained positively for collagen II. Therefore, the superficial area of the meniscus contains a population of cells that can form an articular cartilage-like matrix.

Claims

1. Isolated meniscal superficial zone cells useful for repairing articular cartilage.

2. Isolated meniscal superficial zone cells useful for the promotion of articular cartilage generation.

3. Isolated meniscal superficial zone cells according to claims 1 or 2 that express at least one of the following markers: CD105, Msx-1 , Stro-1 , BST-1 , Fibronectin-EDA, α-smooth muscle actin or superficial zone protein.

4. Isolated meniscal superficial zone cells according to claims 1 or 2 that are derived from meniscal tissue not more than 500μm from the meniscal joint lining surface.

5. Isolated meniscal superficial zone cells according to claims 1 or 2 that are derived from meniscal tissue not more than 150μm from the meniscal joint lining surface.

6. A pharmaceutical composition comprising meniscal superficial zone cells.

7. A pharmaceutical composition according to claim 6 further comprising at least one of the following: saline, buffered solution or hyaluronic acid.

8. A medicament for the treatment of clinical conditions featuring or characterised by articular cartilage deficiency or injury, comprising meniscal superficial zone cells.

9. A medical device comprising meniscal superficial zone cells and a support medium.

10. A medical device according to claim 9 where the support medium is a scaffold.

11. A medical device according to claim 10 where the scaffold is a resorbable or non-resorbable synthetic scaffold.

12. A medical device according to claim 10 where the scaffold comprises collagen, fibrin or hyaluronic acid.

13. A medical device according to claims 10 to 12 where the scaffold is in a fibrous, gel, foam, particulate or sponge format.

14. A medical device according to claim 9 where the support medium is a hydrogel or alginate.

15. A medical device according to claim 9 where the support medium is secreted by the meniscal cells during pellet culture.

16. A medical device according to claim 10 where the scaffold comprises either polyglycolic acid (PGA), polylactic acid (PLA) or co-polymers thereof.

17. A medical device according to claim 10 where the scaffold comprises polyethylene terepthalate (PET) non-woven felt.

18. A method for preventing and/or treating cartilage defects comprising the application of meniscal superficial zone cells to articular cartilage defects.

19. A method according to claim 18 where the cells are administered in an undifferentiated state.

20. A method according to claim 18 where the cells are pre-differentiated prior to administration.

21. A method according to claims 18-20 where the cells are administered in a delivery vehicle.

22. A method according to claim 21 where the delivery vehicle comprises one or more of the following: saline, buffered solutions or hyaluronic acid.

23. A method according to claim 18-22 where the cells are delivered via open surgery.

24. A method according to claim 18-22 where the cells are delivered via intra-articular injection.

25. A method according to claim 18-22 where the cells are delivered arthroscopically.

26. A method of preventing and/or treating cartilage defects comprising the application of a device comprising meniscal superficial zone cells to articular cartilage defects.

27. A method according to claim 26 where the device is delivered via open surgery.

28. A method according to claim 26 where the device is delivered via intra- articular injection.

29. A method according to claim 26 where the device is delivered arthroscopically.

30. A method for the promotion of articular cartilage generation using meniscal superficial zone cells.

31. A method for isolating meniscal superficial zone cells.

32. A method according to claim 31 where the cells are isolated using centrifugal techniques.

33. A method according to claim 32 where the centrifugal techniques are ficol gradients or counter current elutriation.

34. A method according to claim 31 where the cells are isolated using antibody binding.

35. A method according to claim 34 where the antibody binding method is FACS or MACS.

36. A method according to claim 31 where the cells are isolated using selective culture media.

37. A method according to claim 31 where the cells are isolated on the basis of the physical properties of the cell.

38. A method according to claim 37 where the physical property is size or granularity.

39. A method according to claim 31 where the cells are isolated via differential adhesion to a particular substrate.

40. A method according to claim 39 where the substrate is fibronectin.