WO2012001124A1

WO2012001124A1 - Mesenchymal cells and multilayer membrane for the treatment of osteochondral lesions

Info

Publication number: WO2012001124A1
Application number: PCT/EP2011/061051
Authority: WO
Inventors: José Antonio ANDRADES GÓMEZ; José María LÓPEZ-PUERTA GONZÁLEZ; María Dolores CUENCA LÓPEZ; Pedro JIMÉNEZ PALOMO; José BECERRA RATIA
Original assignee: Universidad De Málaga; Ciber-Bbn; Servicio Andaluz De Salud
Priority date: 2010-06-30
Filing date: 2011-06-30
Publication date: 2012-01-05
Also published as: ES2380674A1; ES2380674B1

Abstract

The present invention relates to a pharmaceutical composition comprising mesenchymal stem cells housed in a multilayer membrane that has at least two layers with different structure, with the lower layer being porous and the upper layer being compact, preferably of collagen. Furthermore, the present invention relates to the use of said pharmaceutical composition for the treatment of osteochondral lesions and to the method for the obtainment of said pharmaceutical composition.

Description

MESENCHYMAL CELLS AND MULTILAYER MEMBRANE FOR THE TREATMENT OF OSTEOCHONDRAL LESIONS

The present invention belongs to the field of Biomedicine, Biotechnology, Cell Biology and Regenerative Medicine. The present invention relates to a pharmaceutical composition comprising mesenchymal stem cells housed in a multilayer membrane and to its use for the treatment of osteochondral lesions.

PRIOR ART

Cartilage is a high specialized connective tissue, formed by cells, mainly chrondoblasts or chondrocytes, and fibres, mainly collagen; all embedded in an extracellular matrix (ECM), which is amorphous, viscous, with gel appearance and with a high biochemical, structural and biochemical complexity (Buckwalter JA et al. Arthr Rheumatol, 1998. 41 : 1331 -1342; Johnson, LL. Clin Orthop Rel Res, 2001391 : 306-317).

Articular cartilage (AC) provides the bones on which it is disposed with an elastic and resistant surface, whilst protecting the joint by distributing the pressure (compression) of the load whereto it is subjected. At the same time, together with the synovial fluid, it provides a low friction coefficient that allows the free movement of the joint (Aigner, T. Arthritis Rheum, 2003. 48: 1 166- 1177). The collagen fibres that constitute the ECM of this cartilage are predominantly of type II, accompanied by proteoglycans, responsible, specifically aggrecan, for the high degree of hydration of its matrix, thanks to its capacity for retaining water, around 75%, due to its negative hydrostatic nature. In relation to the cell component, chondrocytes are predominantly round cells, originated from mesenchymal stem cells (MSCs) from the bone marrow (BM) located in matrix cavities (Lin et al., Tissue Eng. 2006. 12(7):1971 -84.), and represent 5-10% of the volume of the solid phase of the cartilage. Chondrocytes are essential in the maintenance of the ECM, regulating both the secretion of its components and the degradation or the remodelling thereof (Hunziker, EB. Osteoarthritis Cartilage, 2002. 16: 564-572). Structurally, AC is divided in three different zones: superficial (SZ), middle (MZ), and deep (DZ). These regions, with different specific functions, are identified by the composition of the ECM, its biosynthesis, the gene expression and the morphology of its cells, as well as the different biomechanical properties thereof (Schinagl, RM et al. J Orthop Res 1997, 15: 499-506; Darling, EM et al. J Orthop Res. 2004, 22: 1182-1 187; Shieh, AC et al. J Biomech. 2006. 39:1595-02).

The great majority of the described chondral lesions occur in the SZ of the cartilage. From a clinical standpoint, the AC has a limited capacity for regeneration, due to: 1 ) its avascular nature, for which reason it does not have a source of circulating stem cells (chondroprogenitor cells; Martin JM et al., Biotechnol Prog, 2005, 21 : 168-177) and the corresponding humoral factor; 2) its aneural nature, with the consequent lack of innervation; and 3) the high complexity of its ECM which, in a possible repair process, does not recapitulate its own development and morphogenesis.

Depending on the depth of the damage, there are three types of lesions that affect the AC: microtrauma, chondral fracture and osteochondral fracture. In any of said situations it is possible to include osteoarthritis (OA), the most common form of arthritis, a highly painful disease, highly prevalent in society (24 million adults in the USA), which is not necessarily a consequence of age, and which directly affects the AC by altering its integrity, limiting the mobility of the joint (Nakamura, N et al. J Arthr Rel Surg, 2009. 25: 531 -55). The aetiology of OA is unknown and is manifested in morphological, biochemical and molecular changes in the cells and in the hyaline cartilage (Christensen, R et al. Osteoarthritis Cartilage, 2005. 13: 20-27).

Currently, the clinical solution of chondral lesions is a challenge for general practitioners and scientists. Repair is a fast process for resolving a lesion, but the reparative tissue is not identical to the original tissue, and there may even be a lack of integration therewith. Regeneration, however, is a relatively slow process that recapitulates the development and morphogenesis of the tissue to treat, completely restoring the structure and function thereof, including a suitable integration with the original tissue. Despite the fact that the treatments of the last years are promising, no process has been found yet that may produce a satisfactory repair or regeneration of the hyaline cartilage and the subchondral bone.

Bearing in mind the capacity of the cartilaginous cell for nourishment by diffusion of the ECM and by synovial imbibing, recently, since the pioneering work of Heatley, FW et al. (J Bone Joint Surg Br. 1980. 62: 397-402) surgical processes have been developed to try to repair the osteochondral defects, or to at least provide a symptomatic relief. The objective is to prevent the extension of the lesions and induce the regeneration of the cartilage, or at least to delay the progression towards OA or the need for joint replacement.

These techniques are divided in 4 categories:

a) symptomatic treatment,

b) stimulation of cells derived from the BM,

c) chondrogenesis with transplanted cells or tissues, and

d) transplants of osteochondral cylinders.

The microfracture (b), which causes the emergence of BM by perforation of the subchondral bone (Buckwalter JA et al. Arthr Rheumatol, 1998. 41 : 1331 - 1342); or the mosaicplasty (d), whether heterologous or autologous, which transfers cylinders of hyaline cartilage from healthy tissue, either in areas with less load close to the lesion, or from another joint (Hunziker et al. Tiss Eng. 2006, 12: 3341 -3364), end up causing the formation of fibrocartilage as cicatricial tissue, which is incapable of restoring the typical characteristics of the AC in the long term, further representing, with the passage of years of the patient, places of easy fracture and new lesions (Buckwalter et al. Clin Orthop Rel Res, 2004. 423: 7-16), besides problems in obtaining the sufficient surface of cartilage to transplant and of damaging one area to repair another. In the case of c), a technique very widespread in clinical practice, the transplant of autologous chondrocytes is being worked on, whether in the absence of a transporter (ACI, Autologous Chondrocytes Implantation,), or carried in a membrane (MACI, Matrix Autologous Chondrocytes Implantation), almost always of collagen. In this sense, some works are focusing on the obtainment of cell cultures of chondrocytes and their implantation in situ, although this process is posing problems due to a lack of take of the implant to the lesion in the medium term. In no case, and to date, is it indicated that ACI/MACI are superior to other therapeutic alternatives in the treatment of knee chondral lesions; rather the contrary, as it is highly invasive and relatively expensive, since it needs two surgical interventions.

Therefore, at present there are no clear criteria as to which of these alternatives is the most suitable for the treatment of AC lesions. With all of this, and in consequence, tissue engineering currently has the challenge of solving the clinical problem for AC regeneration, stimulating the growth of sufficient new cartilage in the place of the lesion and preventing the subsequent loss thereof (Hollander AP et al. Tiss Eng. 2006, 12: 1787-1798).

The cells are unique and irreplaceable effectors of skeletogenesis. Their number and biology suffer significant changes with age and disease, which can be corrected by direct action, inducing their proliferation, migration and differentiation in situ, or indirectly, performing ex vivo processes taking advantage of the knowledge that stem cells and cell therapy provide. The special characteristics of skeletal tissues, where the ECM, as we have seen, reaches special relevance, makes that the carrying of cells and molecules through it allows chondro-osteoconduction and chondro-osteoinduction in the desired manner. The development of chondro-osteoconductor materials, enhanced by the progresses that allow the conquest of nanometric magnitudes, presents a panorama of significant changes in orthopaedic clinical practice. At this time, the experimental strategy is becoming more widespread of trying to search for MSCs susceptible of differentiating into chondroblasts, MSCs which can be isolated from adult tissues, such as BM, skeletal muscle and synovia. The MSCs are attractive for the regenerative medicine of cartilage, as they can be obtained with minimum morbidity, can be easily isolated and expanded in culture and are multipotent, including the chondrogenic lineage (Yoshimura et al. Cell Tiss Res. 2007, 327: 449-462).

Despite this data, there is currently not yet a sole criterion for proposing the most suitable cell therapy, with a clarification of the processes, parameters and conditions in order to select the ideal cell source. DESCRIPTION OF THE INVENTION

The present invention provides an effective product for the cell therapy of articular cartilage (AC) lesions which is prepared in vitro and is based on autologous adult mesenchymal stem cells (MSCs) and a biomaterial with a novel fibrillar organization in the form of a multilayer membrane.

Thanks to this product, a regeneration tissue is formed in the chondro- oesteoarticular lesion identical to the adjacent cartilage in the three cartilaginous strata and identical to the subchondral bone, both in the organization and the distribution of the cells and in the quantity and quality of the cartilaginous or bone surrounding matrix. The regenerated tissue is permanently integrated into the recipient tissue and, furthermore, is functional from the standpoint of its response to load forces.

The inventors of the present invention have demonstrated that this product allows the formation of cartilage naturally and, therefore, that the new tissue is formed de novo and is integrated in the tissue to treat, allowing a stable, lasting and functional regeneration. In other words, this product allows intratissue regeneration more than a repair of the lesion.

The inventors of the present invention present experimental evidence that demonstrates the efficacy of a regenerative therapy which is based on the implant of MSCs capable of differentiating to chondrocytes, transported in a multilayer matrix. The positive results obtained advance in the clinical application of a cell therapy based on MSCs for the treatment of AC lesions.

In the field of Orthopaedic Surgery and Orthopaedics, the present invention provides a product and a methodology for the regeneration of articular hyaline cartilage by means of therapeutic chrondrogenesis and osteogenesis, designed, among others, for patients affected by osteochondral lesions which, in a larger or smaller surface, can trigger localized osteoarthritis and areas of osteochondritis.

The solution offered by the present invention means an improvement to the products and methods known to date.

Therefore, a first aspect of the present invention relates to a composition (hereinafter called composition of the invention) comprising isolated mesenchymal stem cells and a multilayer membrane. Preferably, the source of the mesenchymal stem cells is bone marrow.

Mesenchymal stem cells (MSCs) are multipotent cells, i.e. capable of producing different cell types and capable of for self-renewal, of mesodermal source. MSCs may give rise, both in vitro and in vivo, to adipocytes, chondrocytes, osteoblasts, myocytes, and even to neurones, hepatocytes and pancreatic cells. Bone marrow is a flexible tissue which is found inside some bones, including long bones, the pectoral girdle and the pelvis. Its main function is haematopoiesis, for which purpose it contains hematopoietic stem cells, although it also contains stromal cells, whose function is not directly haematopoiesis, among which the MSCs are found.

It has been extensively described in the art that MSCs can be obtained from different tissues, such as, but not limited to, umbilical cord, bone marrow, adipose tissue, skeletal muscle, synovial tissue, etc. The inventors of the present invention have differentiated MSCs from synovial fluid using the protocol described in Examples 1 and 2 of the present description, and have found that these cells have an excellent proliferative capacity as well as a high chondrogenic potential. MSCs from synovial fluid yield higher chondrogenic differentiation rates than bone marrow derived MSCs. The differentiation protocol has been described in Erickson GR et al. Biochemical and Biophysical Research Communications, 2002. 290, 763-769.

In a preferred embodiment of the first aspect of the present invention, the isolated MSCs are isolated from bone marrow, adipose tissue, skeletal muscle, umbilical cord or synovial tissue, including synovial fluid and synovial membrane. Preferably, they are isolated from synovial tissue.

The MSCs can also be derived from induced pluripotent stem cells. The MSCs can also be derived from embryonic stem cells without the destruction on the embryo. Preferably, the MSCs are adult MSCs. Preferably, the MSCs are not embryonic stem cells.

The term "multilayer", as used in the present description, shall be taken to mean that the membrane has at least two layers each with different structure i.e. comprising of a plurality of structurally distinct layers. Preferably said membrane has two layers and is, therefore, a two-phase membrane.

The term "mesenchymal stem cell" or "MSC" shall be taken to mean a cell isolated from a tissue of mesenchyme origin and capable of differentiating into at least 2,3,4,5 or more cell types.

In a more preferred embodiment of the first aspect of the invention, the

MSCs are positive for or significantly express at least one or more of CD13, CD29, CD44, CD71 , CD90, CD105, CD271 and Strol ; and negative for at least CD34 and CD45. More preferably, the MSCs are positive for one or more of CD13, CD29, CD44, CD71 , CD90, CD105, CD271 and Strol ; and negative for CD34 and CD45.

In a more preferred embodiment of the first aspect of the invention, the MSCs are positive for or significantly express at least CD13, CD29, CD44, CD71 , CD90, CD105, CD271 and Strol ; and negative for at least CD34 and CD45. More preferably, the MSCs are positive for CD13, CD29, CD44, CD71 , CD90, CD105, CD271 and Strol ; and negative for CD34 and CD45.

As used herein, the expression "significantly express" means that, in a cell population comprising the cells of the invention, more than 10%, preferably 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or all of the cells show a signal for a specific cell surface marker in flow cytometry above the background signal using conventional methods and apparatus (for example a Beckman Coulter Epics XL FACS system used with commercially available antibodies and standard protocols known in the art). The background signal is defined as the signal intensity given by a non-specific antibody of the same isotype as the specific antibody used to detect each surface marker in conventional FACS analysis. Thus for a marker to be considered positive the specific signal observed is stronger than 10%, preferably 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 500%, 1000%, 5000%, 10000% or above, than the background signal intensity using conventional methods and apparatus (for example a Beckman Coulter Epics XL FACS system used with commercially available antibodies and standard protocols known in the art).

The CD markers make reference to molecules that are found on the cell surface and which can be detected with specific antibodies. Their name comes from "cluster of differentiation" and they are commonly used for the identification of the cell type, the differentiation stage or the cell activity. Strol relates to an antibody which has been described as capable of recognizing bone marrow MSCs.

In a preferred embodiment of the first aspect of the invention, per each mm² of membrane there is between 250,000 and 2,000,000 isolated MSCs. Preferably, per each mm² of membrane there is between 400,000 and 1 ,000,000 isolated MSCs. More preferably, per mm² of membrane there is between 450,000 and 600,000 isolated MSCs.

In a preferred embodiment of the first aspect of the invention, the multilayer membrane comprises at least one compact layer with pores of a diameter smaller than 35 μηι and at least one porous layer comprising pores of a diameter between 35 and 300 μηπ.

The multilayer membrane of the present invention may be made of many different materials, as there are many biomaterials useful for the preparation of a multilayer membrane with controlled pore size in each layer. These materials are well known by a skilled in the art. Without limiting the present invention, some examples of these materials are collagen, poly-L-lactic acid (PLLA) and chitosan. Preferably, the multilayer membrane comprises collagen. More preferably, the collagen is of type I and III.

The multilayer membrane of the present invention preferably comprises any of the following types of collagen: I, II, IX, XI, VI, X, XII and XIV. More preferably, it comprises cartilage-specific collagens, like type II, IX and/or XI.

Collagen is a protein characteristic of connective tissue, where it is secreted to the extracellular matrix (ECM) by the cells that form it. The collagen is aggregated and forms fibres of around 300 nm in length and 1 .5 nm diameter. To date, 29 types of collagen have been described, although types I, II, II and IV are most abundant. Type I is the most abundant and is found in the bones, the skin, the tendons and the arterial walls. It forms fibrils which are grouped in fibres and gives resistance to the tissue. Type III is the main component of the cross-linked fibres and is more abundant in the loose conjunctive tissue, in the blood vessel walls, in the intestine and uterus. It is associated to the collagen I fibrils and acts as support in expandable tissues.

In preferred embodiment of the first aspect of the invention, the membrane comprises glycosaminoglycans and/or proteoglycans that form the ECM of the hyaline cartilage. The preferred glycosaminoglycans are chondroitin sulphate, keratan sulphate, heparan sulphate and/or hyaluronic acid. The preferred proteoglycan is aggrecan.

In the compact layer, the density of collagen fibres is so high that there are barely cavities left between them which can house the isolated MSCs. It is a layer with mechanical resistance similar to that of the cartilage. In the porous layer of the membrane there are less collagen fibres and they are disposed so that they leave large spaces between them capable of housing the isolated MSCs.

In a preferred embodiment of the first aspect of the invention, the pores of the porous layer have between 4 and 14 million μηπ³ of average volume.

In another preferred embodiment of the first aspect of the invention, between 70% and 90% of the cells are located in the porous layer of the membrane.

In another preferred embodiment of the first aspect of the invention, the membrane is coated with a composition which favours cell adhesion. This composition may include any molecule which favours cell adhesion, such as some proteins of the extracellular matrix or some polymers such as polylysine or polyornithine.

In another preferred embodiment of the first aspect of the invention, the membrane is coated with a molecule of the extracellular matrix. Preferably, the molecule of the extracellular matrix is fibronectin, laminin or collagen. More preferably, it is fibronectin.

Fibronectin is a glycoprotein typical of the ECM involved in processes such as cell adhesion, migration, growth and differentiation.

In a preferred embodiment of the first aspect of the invention, the membrane imitates the form of an osteochondral lesion and has dimensions equal to or greater than those of the lesion, so that the membrane, in position superimposed on the lesion, protrudes with respect to said lesion in a magnitude less than 2 mm.

In a preferred embodiment of the first aspect of the invention, the composition is obtained by a process comprising the following steps:

a) obtaining an isolated sample from a subject,

b) culturing the mesenchymal stem cells of the sample obtained in step (a),

c) introducing the mesenchymal stem cells cultured in step (b) in the multilayer membrane by vacuum.

In a preferred embodiment of the first aspect of the invention, the composition is a pharmaceutical composition.

The pharmaceutical composition of the invention can be designed as an implant whose dimensions are adapted to the size of the lesion in question. Generally, the articular cartilage is a thin tissue, with a depth of around 1 .5 cm in humans and at least 3 mm in rabbits. The dimensions of the membrane adapt, therefore, to the surface of the lesion and can be such that each side is 1 mm larger than the lesion surface. For example, for a lesion of 3 mm in length, 4 mm wide and 3 mm deep, a membrane can be prepared of 4 mm long and 5 mm wide. Once the membrane is ready, coated or not, the MSCs would be introduced therein.

To place the membrane with the MSCs in the lesion, it is sufficient to introduce said membrane in the cavity left by the lesion, lightly compressing the membrane so that it enters in said lesion. The slightly larger size of the membrane allows it to remain in the desired position and it does not move nor come out when the joint is used. In this way, it avoids the suture of the membrane to the lesion, which speeds up the implant process and does so more simply, although in cases of large lesions it may be necessary to use suturing to place the membrane with the MSCs in the lesion.

In another preferred embodiment of the first aspect of the invention, the pharmaceutical composition furthermore comprises a pharmaceutically acceptable excipient.

An excipient is a component of a pharmaceutical composition which is not an active compound but a diluent, a vehicle or filler, among others, which is considered pharmaceutically acceptable when it is safe, non-toxic and does not present adverse effects.

In another preferred embodiment of the first aspect of the invention, the pharmaceutical composition furthermore comprises another active principle.

A second aspect of the present invention relates to the use of the pharmaceutical composition of the invention as a medicament. Preferably, the pharmaceutical composition is used for the treatment of a cartilage lesion. More preferably, for cartilage regeneration. Preferably, the pharmaceutical composition is used for the treatment of a bone lesion. More preferably, it is for bone regeneration. More preferably, it is used for the treatment of an osteochondral lesion. Even more preferably, the pharmaceutical composition is used for the treatment of osteoarthritis. An osteochondral lesion is a lesion of the cartilage and the subchondral bone. Osteoarthritis is a disease in which a wear of the cartilage occurs, which causes the friction of the bones and their subsequent wear or inflammation.

The pharmaceutical composition of the present invention can be used as an implant in the regenerative therapy of cartilage lesions. This pharmaceutical composition can be administered by placing it in the chondral lesion area so that the compact layer of the multilayer membrane is oriented towards the synovial fluid and the porous layer, which contains the greater part of the isolated MSCs, is oriented towards the injured tissue.

As shown in example 3 and in figure 9 of the present description, the inventors demonstrate that the pharmaceutical composition of the present invention implanted in an osteochondral lesion, is capable of regenerating the injured tissue, both the cartilage and the subchondral bone, leaving the new tissue perfectly integrated with the healthy tissue and having a normal histological structure.

A third aspect of the present invention relates to a method for the obtainment of the pharmaceutical composition of the invention comprising:

a.- obtaining an isolated sample from a subject,

b.- culturing the MSCs of the sample obtained in step (a),

c- introducing the MSCs cultured in (b) in a multilayer membrane by vacuum.

In a preferred embodiment of the third aspect of the invention, the sample is isolated from synovial tissue, adipose tissue, skeletal muscle tissue, umbilical cord or bone marrow. Preferably, the sample is isolated from bone marrow.

The MSCs are isolated by culturing the isolated sample obtained in step

(a).

For example, the samples of bone marrow (BM) are isolated by puncture and extraction of a small quantity of blood from the bone, which receives the name of BM aspirate, or from a small cylinder of bone, which receives the name of BM biopsy. The aspirations are usually performed in the sternum or in the posterior iliac crest, whilst the biopsies are usually performed in the anterior or posterior iliac crest.

The MSCs can be cultured as described in example 1 of the present description. The introduction of the isolated MSCs in the membrane can be performed as described by Solchaga LA et al. Tissue Eng. 2006 12(7): 1851— 1863.

In a chondral or osteochondral lesion, the multilayer membrane can be placed with the compact layer towards the synovial fluid and the porous layer towards the injured tissue. In this way, the most compact and dense layer, which barely houses isolated MSCs, prevents them from exiting the membrane but allows them to be communicated with said synovial fluid, since the compact layer does not block substance exchange. The isolated MSCs located in the porous layer are oriented towards the lesion and, on regenerating new cartilage and/or bone tissue they may be incorporated in the non-injured tissue so that the new tissue is perfectly integrated.

In a preferred embodiment of the third aspect of the invention, the multilayer membrane of step (c) is coated with a composition which favours cell adhesion.

In a preferred embodiment of the third aspect of the invention, the multilayer membrane of step (c) is coated with a molecule of the extracellular matrix (ECM). Preferably, the molecule of the ECM is fibronectin or laminin. More preferably, the molecule of the ECM is the fibronectin.

ECM is the extracellular or interstitial medium of a tissue. Its nature is complex and its composition different in each type of tissue, although in animals it is composed largely of glycoproteins and glycosaminoglycans. Its main functions are mechanical resistance and nutritional support.

An embodiment of the second aspect of the invention is the use of a multilayer membrane with a compact layer and another porous layer, of collagen I of 3 mm wide by 3 mm long where the pores of the porous layer have a volume of 6 million μηπ³ and where 5.4 million isolated MSCs are introduced from a BM culture of an iliac crest aspirate of an individual, for the treatment of an osteochondral lesion in said individual.

An embodiment of the second aspect of the invention is the use of a multilayer membrane with one compact layer and another porous layer, of collagen II of 4 mm wide by 6 mm long, coated with laminin, where 4.8 million isolated BM MSCs are introduced by vacuum, for use as a medicament.

An embodiment of the second aspect of the invention is the use of a two- phase membrane with one compact layer and another porous layer, comprising collagen I and II of 5 mm wide by 10 mm long, coated with a biocompatible fibrous material, for example, but without being limited to, fibronectin, where the pores of the porous layer have an average volume of 7 μηπ³ and where 30 million isolated MSCs are introduced by vacuum.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For persons skilled in the art, other objects, advantages and characteristics of the invention will be inferred in part from the description and in part from the practice of the invention. The following figures and examples are provided by way of illustration, and are not intended to limit the present invention.

DESCRIPTION OF THE FIGURES

Figure 1. Shows an in vitro culture of adult mesenchymal stem cells

(MSCs) in culture passage 0 (pO) showing its characteristic fibroblastic aspect, 7 days after its isolation from a bone marrow aspirate from the iliac crest of a rabbit. Image obtained by inverted optical microscopy.

Figure 2. Shows a confluent in vitro culture of adult mesenchymal stem cells (MSCs) in culture passage 1 (p1 ), 12 days after its isolation from a bone marrow aspirate from the iliac crest of a rabbit. Image obtained by inverted optical microscopy.

Figure 3. Shows an in vitro culture of adult mesenchymal stem cells (MSCs) in culture passage 1 (p1 ), predifferentiated to the cartilaginous lineage in the presence of a chondroinductor culture medium during 12 days. Image obtained by inverted optical microscopy.

Figure 4. Shows the multilayer membrane and the mesenchymal stem cells (MSCs) therein, (a) Side where the collagen fibres are distributed in more loosely manner, which is placed towards the lesion, (b) More compact side which is placed towards the synovial space, (c) Shows the number and the distribution of the MSCs in the loose side, (d) Shows the number and the distribution of the MSCs in the compact side. Images obtained by electronic scanning microscopy.

Figure 5. Shows the femoral condyle of the rabbit knee where a complete osteochondral defect has been performed (3x3 mm) in the maximum load zone.

Figure 6. Represents the osteochondral defect in the femoral condyle of the rabbit knee at the time of placing the implant, time 0 (tO). The arrows indicate the membrane with the cells, (a) Shows the size of the implant, (b) Shows the introduction of the implant inside the lesion.

Figure 7. Shows the result of the untreated osteochondral lesion after 1 and 6 months, where it is possible to observe that the defect remains without regenerating, as it is a lesion that does not regenerate by itself, (a) Shows a lesion in the femoral condyle of the rabbit knee 1 month after surgery, (b) Shows a lesion in the femoral condyle of the rabbit knee 6 months after surgery, (c) Shows the histological result of the lesion area 1 month after surgery, (d) Shows the histological result of the lesion area 6 months after surgery. In (c) and (d) only the formation of fibrous tissue is observed and the complete lack of any sign of cartilaginous matrix. Figure 8. Shows the result of the osteochondral lesion treated with predifferentiated autologous MSCs in vitro towards the chondrogenic line after 6 months, where it can be observed that the defect is coated by a whitish tissue and that the edges of the tissue formed are not welded together with the healthy surrounding cartilage, (a) Shows an osteochondral lesion in the femoral condyle of the rabbit knee 6 months after surgery, (b) Shows the histological result where the formation of a fibrocartilage is observed with a poor formation of cartilaginous matrix, whilst confirming that the edges of the lesion are not welded together with the healthy lateral cartilage (darker, in the left lateral).

Figure 9. Shows the result of an osteochondral lesion in the femoral condyle of the rabbit knee, treated with undifferentiated autologous MSCs in vitro (culture passage p1 ), 6 months after surgery, (a) It is observed that the lesion has a shiny appearance, it appears completely coated with tissue integrated in the edges of the healthy surrounding tissue, (b) Detail of the lesion area, (c) Shows the histological results where the complete regeneration of the osteochondral tissue is observed, with a typical hyaline cartilaginous matrix, with normal thickness, as well as a total remodelling of the underlying subchondral bone, which reaches the natural height until contact with the inferior cartilaginous side.

EXAMPLES

Below, the invention is illustrated by tests performed by the inventors, which reveal the efficacy of the implant comprising MSCs in a multilayer membrane for the regeneration of osteochondral lesions.

EXAMPLE 1 : in vitro processing: obtainment and preparation of the cells.

The experiments are carried out using 40 rabbits of the New Zealand variety, of around 3 kg in weight, in accordance with the proceedings of the Ethics Committee of the University of Malaga on the treatment of experimental animals.

The obtainment of the tissues, as well as the transplant operations, were carried out under anaesthesia with buprenorphine (0.05 mg/kg), ketamine (25- 35 mg/kg) and xylazine (5-10 mg/kg), administered intramuscularly. The two knees were injured, for which purpose, in the case of implants, one knee served as control of the other.

The bone marrow (BM) was obtained by puncture and aspiration in the iliac crest using a flexible catheter and an 18G needle. Around 4 ml of BM were obtained in the presence of 1 ml of heparin. The cells obtained were washed twice in non-supplemented culture medium, they were centrifuged and were resuspended in complete culture medium (DMEM, "Dulbecco's Modified Eagle Medium", 10% FCS, "Fetal Calf Serum", 1 .5% L-glutamine, 1 % Penicillin/Streptomycin and 0.5% amphotericin B) and they were cultured in 75 cm² bottles (Nunc Int., USA) at 37°C and 5% C0₂.

After 4 days, the first change of medium was performed to eliminate the non-adhered cells and, from here, the changes of medium were performed every 3 days until a total of 14 days at culture passage 0 (pO). Then, the cells were trypsinized, counted and seeded at culture passage 1 (p1 ) in 60 cm² culture plates, at a density of 10, 50 and 100 cells/cm². At this time, several frozen vials were kept for later study, using 5% DMSO in complete medium and at a density of 1x10⁶ cells/ml.

After another 7 and 14 days in culture, the cells were collected from each cell density well and they were counted in a hemocytometer, in order to calculate the rate of growth. A routine flow cytometry was performed to certify the phenotypical profile of the cells obtained, showing that the BM MSCs isolated are positive for CD13, CD29, CD44, CD71 , CD90, CD105, CD271 and Strol ; and negative for CD34 and CD45.

EXAMPLE 2: Verification of the chondrogenic potential of the BM MSCs.

To induce chondrogenic differentiation, 250,000 cells in culture passage

1 (p1 ) were taken to a 15 ml propylene tube (Becton Dickinson) and they were centrifuged at 450 g during 10 minutes. The cell precipitates were cultured during 12 days in chondroinductor medium, composed of DMEM with 10 ng/ml TGF-beta1 (R&D Systems), 100 nM dexamethasone. 50 micrograms/ml ascorbic acid, 100 micrograms/ml sodium pyruvate, 40 micrograms/ml proline (Sigma), and ITS-plus (6.25 micrograms/ml bovine insulin, 6.25 micrograms/ml transferrin, 6.25 micrograms/ml selenic acid, 5,35 micrograms/ml linoleic acid, 1.25 micrograms/ml bovine serum albumin; Collaborative Biomedical Products).

In order to evaluate the chondrogenic differentiation, the cell pellets were processed for microscopic analysis in paraffin for conventional histology. The sections in paraffin were processed for staining with alcian blue, which specifically marks the proteogylcans of the cartilaginous matrix, and for the immunohistochemistry against collagens I, II and X. The marking for collagen I was positive in the regenerated bone, whilst positive marking of collagens II and X were found in the new cartilage.

Furthermore, the gene expression of collagens I, II and X, of Sox9 and of aggrecan were analysed by RT-PCR.

EXAMPLE 3: Osteochondral lesion and implant.

After the analgesia or anaesthesia of the animals, as indicated above, we performed the shaving of the two knees and the disinfection of the surgical field.

We tackled the joint by medial parapatellar incision and rejection of the patella towards the exterior, to then cause an osteochondral lesion in the femoral condyle in the load zone, with a 3x3 mm trocar. Lesions of this size exclude spontaneous repair. The defect was washed with physiological serum and the collagenic membrane was implanted in the right knee experimental situation, previously coated with fibronectin, adsorbed by vacuum with 2x10⁶ previously obtained BM MSCs.

The cell transporter has dimensions of 2 x 2 x 1 mm and is formed by collagen I and III fibres disposed so that, in the upper surface (surface placed towards the synovial space), appears more densely interlinked, and in the lower part, which is placed towards the walls and floor of the osteochondral lesion, the fibres appear more relaxed leaving an average space of 250 μηπ, where 83% of the adsorbed cells reside. This biomaterial is commercial (Chondro-Gide®, Geistlich Pharma, Switzerland) and is being widely used in clinical knee surgery. The implant is carried out without any type of adhesive or suture, eliminating any type of interference in the study, reducing the morbidity and the time of intervention.

The control knee is implanted with the membrane without cells and the surgery is carried out in the same way. Finally, both wounds are sutured, and analgesic and antibiotics are given subcutaneously to the animal during 5 days post-surgery. The rabbits are sacrificed by an overdose of nembutal 4, 12, and 24 weeks after surgery, in order to assess the results of the regeneration, by a macroscopic and histological examination of the lesion, following the typical parameters (Wakitani evaluation).

Claims

1. Composition comprising isolated mesenchymal stem cells and a multilayer membrane that has at least two layers with different structure, with the lower layer being porous and the upper layer compact.

2. Composition according to the preceding claim, wherein the isolated mesenchymal stem cells are positive for at least CD13, CD29, CD44, CD71 , CD90, CD105, CD271 and Strol ; and negative for at least CD34 and CD45.

3. Composition according to the preceding claim, wherein the isolated mesenchymal stem cells are positive for CD13, CD29, CD44, CD71 , CD90, CD105, CD271 and Strol ; and negative for CD34 and CD45.

4. Composition according to any of the preceding claims, wherein per each mm² of membrane there are between 250,000 and 2,000,000 cells.

5. Composition according to the preceding claim, wherein per each mm² of membrane there are between 400,000 and 1 ,000,000 cells.

6. Composition according to any of the two preceding claims, wherein per each mm² of membrane there is between 450,000 and 600,000 cells.

7. Composition according to any of the preceding claims, wherein the multilayer membrane comprises at least one compact layer comprising pores the diameter whereof is less than 35 μηι and a porous layer comprising pores the diameter whereof is between 35 and 300 μηπ.

8. Composition according to the preceding claim, wherein the pores of the porous layer have between 4 and 14 million μηπ³ of average volume.

9. Composition according to any of the preceding claims, wherein the multilayer membrane comprises collagen, poly-L-lactic acid (PLLA) and/or chitosan.

10. Composition according to the preceding claim, wherein the multilayer membrane comprises collagen.

11 . Composition according to the preceding claim, wherein the collagen is of type I and III.

12. Composition according to claim 10, wherein the collagen is of type I, II, IX, XI, VI, X, XII and/or XIV.

13. Composition according to the preceding claim, wherein the collagen is of type II, IX and/or XI.

14. Composition according to any of the preceding claims, wherein the multilayer membrane comprises glycosaminoglycans and/or proteoglycans.

15. Composition according to the preceding claim, wherein the glycosaminoglycans are selected from chondroitin sulphate, keratan sulphate, heparan sulphate and/or hyaluronic acid and the proteoglycan is aggrecan.

16. Composition according to any of the preceding claims, wherein between 70% and 90% of the cells are located in the porous layer of the membrane.

17. Composition according to any of the preceding claims, wherein the membrane is coated with a molecule of the extracellular matrix.

18. Composition according to the preceding claim, wherein the molecule of the extracellular matrix is fibronectin, laminin or collagen.

19. Composition according to the preceding claim, wherein the molecule of the extracellular matrix is fibronectin.

20. Composition according any of the preceding claims, wherein the membrane imitates the form of an osteochondral lesion and has dimensions equal to or greater than those of the lesion, so that the membrane, in position superimposed on the lesion, protrudes with respect to said lesion in a magnitude less than 2 mm.

21 . Composition according to any of the preceding claims, wherein the isolated mesenchymal stem cells are isolated from synovial tissue, adipose tissue, skeletal muscle tissue, umbilical cord or bone marrow.

22. Composition according to the preceding claim wherein the isolated mesenchymal stem cells are isolated from bone marrow.

23. Composition according to claim 21 wherein the isolated mesenchymal stem cells are isolated from synovial tissue.

24. Composition according to any of the preceding claims, obtainable by a process comprising the following steps:

a) obtaining an isolated sample from a subject,

b) culturing the mesenchymal stem cells of the sample obtained in step (a),

25. Composition according to any of the preceding claims, wherein said composition is a pharmaceutical composition.

26. Composition according to the preceding claim, further comprising pharmaceutically acceptable excipient.

27. Composition according to any of the two preceding claims, further comprising another active principle.

28. Composition according to any of the preceding claims, for use as a medicament.

29. Composition according to any of claims 1 to 27 for use in the treatment of a cartilage lesion.

30. Composition according to any of claims 1 to 27 for use in cartilage regeneration.

31 . Composition according to any of claims 1 to 27 for use in the treatment of a bone lesion.

32. Composition according to any of claims 1 to 27 for use in bone regeneration.

33. Composition according to any of claims 1 to 27 for use in the treatment of an osteochondral lesion.

34. Composition according to any of claims 1 to 27 for use in the treatment of osteoarthritis.

35. Method for the obtainment of the composition according to any of claims 1 to 27, comprising the following steps:

a) obtaining an isolated sample from a subject,

b) culturing the mesenchymal stem cells of the sample obtained in step (a),

c) introducing the mesenchymal stem cells cultured in step (b) in a multilayer membrane by vacuum.

36. Method for the obtainment of the composition according to the preceding claim, wherein the sample is isolated from synovial tissue, adipose tissue, skeletal muscle tissue, umbilical cord or bone marrow.

37. Method for the obtainment of the composition according to the preceding claim, wherein the sample is isolated from bone marrow.