WO2014041516A1

WO2014041516A1 - Biocompatible multi-layered structure comprising foam layers and a functional interface

Info

Publication number: WO2014041516A1
Application number: PCT/IB2013/058531
Authority: WO
Inventors: Mathieu CUENOUD; Pierre-Etienne Bourban; Jan-Anders Edvin Manson
Original assignee: Ecole Polytechnique Federale De Lausanne (Epfl)
Priority date: 2012-09-14
Filing date: 2013-09-13
Publication date: 2014-03-20

Abstract

A biocompatible multi-layered structure comprising a first foam layer showing a first degree of porosity and a second foam layer showing a second degree of porosity, both foam layers being attached to one another through an interface, wherein said interface is obtained from at least one of said foam layers and has a degree of porosity which is less than the degree of porosity of any of said foam layers.

Description

Biocompatible multi-layered structure comprising foam layers and a functional interface

FIELD OF THE INVENTION

This invention relates to cellular products based on polymers and composites, which integrate at least one interface and the method to process them. The integrated interfacial membrane preferably provides a selective permeability and additional stiffness to the cellular products. Such structures may advantageously be used in: tissue engineering as implantable devices for bone, cartilage and soft tissue replacement, as well as in consumer goods, transportation or in any other suitable field.

BACKGROUND Cellular materials

Cellular materials are widespread in nature (bone, wood, sponge....) as well as in synthetic materials (foams, honeycomb for sandwich structures...) (Gibson, Ashby et al. 2010). For instance, bone is a foam-like cellular material. It is a composite of natural ceramic and polymer materials with different distribution of porosity and mechanical properties. The bone structure offers performance in terms of lightness, stiffness, strength, shapes, porosity, healing performance etc. Thus, polymer foams have been developed to mimic the bone structure for use as scaffold for the regeneration of the bone tissue (WO 2008/007332 Al). Mechanical properties of these cellular solids mainly depend on the morphology of the pores, the properties of the raw material and the density of the foam (Gibson and Ashby 1988). Several foaming processes have been developed according to the different polymer types to obtain foams with various properties. Solvent casting and leaching, gas foaming, emulsion freeze- drying and thermally induced phase separation are the main techniques of foaming that have been developed with their own advantages and drawbacks. They can be adapted to thermoplastics and thermoplastic -based composites, and more specifically to bioresorbable and biocompatible polymers. Composite materials

Polymer composites are well known for their high intrinsic stiffness, lightness, unique combinations of properties and design concepts. Composites based on different polymer matrices and various types of reinforcements such as fibers and fillers offer a wide range of properties and functions. Composition, orientation and architecture of the reinforcement can be adjusted to tailor the final performance. Polymer based composites have been proposed for many applications in the transportation, sport and mechanical engineering industries (Manson, Wakeman et al. 2000). Composites based on polymers are currently emerging as the next generation of materials for applications in the biomedical field. They offer unique design opportunities, better specific mechanical properties and less brittleness than the current metallic- and ceramic-based materials. Furthermore, they can be tailored to act as drug delivery systems.

Multi-layered structures

The assembling of layers to obtained multi-layered structures has been widely studied in composite material science. The sandwich- structured composite, for instance, is fabricated by assembling two external stiff skins to a lightweight but thick internal core. The core material is usually a low strength material (open or closed cell foams, honeycomb structures...) and the skins are laminates (glass or carbon fibers reinforces thermoplastics or thermosets). The sandwich structures provide structural parts of high bending stiffness with an overall low density.

In nature, bone is also a lightweight structure composed of highly porous open cell core (called trabecular bone) surrounded by a hard outer layer (called cortical bone) of low porosity. Cartilage also has a multi-layered structure with layers of different composition and collagen fibers orientation. More generally, the osteochondral tissue, composed of bone and cartilage tissue is a multi-layered tissue with a relative stiff part, the bone, and a relative soft part, the cartilage. Generally, for multilayered structure, if the bond between the layers is to weak, the most probable results will be delamination. The adhesive between the core and the skins in sandwich structures should be strong enough. For the osteochondral tissue, it is the calcified cartilage zone that ensures a strong bond between the subchondral bone and the cartilage. Scaffold design

Scaffolds for tissue engineering provides a guide for the proliferation and migration of cells into the matrix and gives mechanical stability. The material is ideally biocompatible and bioresorbable, and the degradation rate has to match with the extracellular matrix production. Three dimensional (3D) porous scaffolds or hydrogels are the most widespread solutions developed for tissue engineering constructs. The scaffold macrostructure is an important parameters acting on the resulting mechanical properties as well as the proliferation and migration of cells into the scaffold (Cao, Ho et al. 2003; Mano and Reis 2007). The mechanical properties should ideally match those of the repaired tissue and be able to bear load.

The scaffold structure should be highly porous to provide an environment with a large surface-to-volume ratio into which cells could proliferate and express their own extracellular matrix (Butler, Goldstein et al. 2000). The presence of fully interconnected channels is essential to enhance the diffusion of nutrients to the center of the scaffold and the transport of waste products away from it. Open-cell foams offer such features. The optimal size of interconnections of scaffold depends of the tissues to regenerate. For example, in the case of bone repair, the pore size should be large enough to allow ingrowth of blood vessels as bone formation needs vascular support and blood circulation ensures then nutrition of bone (Cao, Ho et al. 2003). The scaffold should therefore allow a rapid development of vascularization. The minimum pore diameter required for ingrowth of cells into the interior of the matrices is 100 μιη (Laurencin, Attawia et al. 1996) and it has been suggested that the ideal pore-size range of 200-400 μιη is preferred by osteoblasts because it provides the optimum compression and tension on the osteoblast's mechanoreceptor (Boyan, Hummert et al. 1996). On the other hand, cartilage is normally fed by articular fluid, and a cartilage scaffold should therefore prevent the formation of blood vessel. Scaffolds for bone repair should ideally be osteoactive: i.e. (i) osteoconductive, to induce cell differentiation into bone cells and (ii) osteoinductive, to enable the growth of bone within the scaffold. Finally, the material should be easily processed into complex shaped components and be easy to sterilize. Polymers and composites in biomedical applications

The materials reported in literature for bone and cartilage scaffolds, are ceramics, polymers, composite materials and bioactive molecules.

The most commonly used ceramics as biomaterials for tissue engineering are calcium phosphate (Ca-P) such as hydroxyapatite (HA) and β-tricalcium phosphate (TCP), or bioactive glasses (such as Bioglass®). Current ceramic implants have an intrinsic low toughness and cannot easily be shaped or screwed; that is why they are not considered as ideal. Biocompatible and bioresorbable polymers present instead ductile properties. Natural or synthetic polymers are widely used for bone and cartilage scaffold. Natural derived polymers such as proteins, especially from extracellular matrices (e.g. collagen or glycosaminoglycan GAG), polypeptides, polysaccharides (including chitosan, starch, hyaluronic acid and alginate) and poly(hydroxylkanoates) have been used because they are bioactive i.e. they usually contain domains that can send important signals to guide cells at various stages of their development. Nevertheless such bioactivity can cause problems with antigenicity and the processing of these materials is often difficult (Mano and Reis 2007).

The most popular biodegradable synthetic polymers include poly(cc-hydroxy acids), especially poly(lactic acid) (PLA), poly(glycolic acid) (PGA) and their co-polymer (PLGA), poly(e- caprolactone), poly(propylene fumarate), poly(dioxanone), polyorthoesters, polycarbonates, polyanhydrides and polyphosphazenes (Mano and Reis 2007). These polymers offer a wide range of chemical, processability variety, and can be obtained with controlled distribution of molecular weights. A wide range of mechanical properties and resorption rates can thus be considered. Nevertheless, some synthetic polymers present limitations such as the production of residues of degraded materials, which can change the microenvironment of the implantation site and somewhat limit the cellular proliferation and tissue formation (Guo, Wang et al. 2004).

Synthetic polymers have frequently been chosen for tissue engineering applications because their degradation can be tailored, however in a highly porous configuration their mechanical properties may be limited. Their mechanical properties can be improved by addition of fibers or particles (fillers) to obtain a composite material. For example, polylactides-co-glycolides have been reinforced by incorporation of polyglycolic acid fibers. It has been demonstrated that the mechanical properties were improved during the initial weeks of healing (Niederauer, Slivka et al. 2000). Ceramic fillers of hydroxyapatite or β-tricalcium phosphate have also been incorporated in poly(L-lactic acid) bioresorbable foams improving considerably the foam stiffness (Mathieu 2004; Mathieu, Montjovent et al. 2005; Mathieu, Mueller et al. 2006) (WO 2008/007332 Al). Moreover, the addition of a bioactive agent such as calcium sulfate or Bioglass in the bone phase may have a beneficial effect on the overall osteochondral healing (Niederauer, Slivka et al. 2000).

Membranes in biomedical implants

Membrane implants are used in surgery to provided barrier layer to molecular substances, cells or vessels. For example, patent application W09919005 describes a multi-layer collagen material that acts as a membrane to provide an impermeable barrier, which guide tissue regeneration and inhibits undesired cell growth of any surrounding connective tissue. This membrane may be adhered to a graft with an adhesive, with pins or sutures. Another example of barrier is given in Patent WO2007046746, which describes a biodegradable oestochondral implant that comprised two porous layers separated by a barrier. This barrier is impermeable to agents that have a detrimental effects on the cartilage regeneration such as blood and other cells, and also to molecules of 5000 to even 100,000 Dalton or more. The membrane isolates the cartilage part from contact with blood located in the subchondral bone part.

Assembling of layers

Biomedical applications may require the assembling of different layers to create a multi- layered structure. For instance, several scaffolds have been developed to mimic the structure of the osteochondral tissue, which contains bone and cartilage tissues. Among the different scaffolds currently proposed as osteochondral graft, the layers have been assembled via suturing (Schaefer, Martin et al. 2000; Schaefer, Martin et al. 2002), gluing (Niederauer, Slivka et al. 2000; Gao, Dennis et al. 2001) and infiltration (Hung, Lima et al. 2003). The addition of an adhesive material may restrict the cell movement and nutrient diffusion across the interface and the use of suturing is often associated with the creation of a void between the layers that can be detrimental to the migration of living cells from the bone to the cartilage scaffold (Harley, Lynn et al. 2010). Furthermore, the mechanical stability of the interface has received little attention in the literature. Delamination of the interface between two layers of different composition and structure may occur due to the stress concentration resulting from an abrupt change in physical properties (elasticity, swelling, etc.) at the interface (Sherwood, Riley et al. 2002). Delamination due to faster resorption of the adhesive, which leads to weak mechanical strength of the interface, may also occur. To prevent delamination, the development of a continuous interface has therefore been investigated. For example, an osteochondral graft based on poly(glycolic acid) and polylactide with a gradient of composition and porosity was obtained using a three-dimensional printing process (Sherwood, Riley et al. 2002). A gradual and continuous interface was also observed in scaffolds produced by (i) interdiffusion of two layered suspensions placed in contact, which results in a gradual change in materials composition at the interface, (ii) followed by solidification using freeze-drying. This scaffold showed no signs of delamination following repeated cycles of loading (Harley, Lynn et al. 2010). As example of assembling methods, we can mention patent application WO02070030 that describes a material assembled with the aid of glue, suture or items such as screws or nails.

Clinical need

Articular cartilage is a soft tissue that covers the ends of articulating bones, and is essential for maintaining joint motion, i.e. translation and rotation between the bones. Articular cartilage nevertheless has a limited capacity for self-repair, and cartilage lesions following traumatic injury may consequently initiate an irreversible degenerative process. This ultimately leads to progressive loss of joint function, pain and medical costs. The loss of osteochondral tissue thus requires reconstructive surgery to repair the defect. Many different clinical treatments have been investigated, but these have not so far led to satisfactory long- term solutions. Tissue engineering research has therefore focused on the preparation of synthetic bioresorbable scaffolds that provide a template for cells and guide the process of tissue regeneration.

In summary, there is a need for a scaffold that will permit successful ingrowth of bone and cartilage tissue, in respective parts of the scaffolds, following implantation in vivo. Therefore, scaffolds should integrate multi-functional membranes: (i) to prevent undesired ingrowth of bone in the cartilage part, (ii) to act as barrier for blood vessel and transport of species and (Hi) to improve the mechanical properties. There is actually a need for new synthetic scaffolds integrating a functional membrane acting as a barrier and stiffening part, which can be used in different surgical situations, especially for load-bearing applications and multi-tissue application. Scaffolds based on porous polymer composites represent then an attractive solution. By assembling different foam layers and controlling the interface inbetween the layers, structures can be tailored which are similar to the natural osteochondral architecture, having each layers with properties close to those of the native bone and cartilage joined by a continuous interface that (i) ensure strong mechanical bonding between the layers, (ii) act as a barrier (fluids, cells, vessels) from one layer to the other and (Hi) provide enhanced stiffening and mechanical stability of the overall structure.

Processing such a multi-layered structure incorporating a functional membrane with a simple and solvent free method is an appealing challenge. A unique solution was validated and is proposed hereafter.

GENERAL DESCRIPTION OF THE INVENTION

The present invention relates to a biocompatible multi-layered structure and related process as defined in the claims. The present invention advantageously relates to a multi-layered cellular structure integrating at least one interface with enhanced and tailored barrier and mechanical properties. The cellular structure incorporates at least two layers and an interface, which creates a multi- layered structure. The cellular layers are advantageously composed of one or more thermoplastic polymers or composites. The process can combine different types of cellular layers with different porosities, mechanical properties and polymer compositions in order to tailor the properties of each layer of the product. The obtained interface properties, in terms of barrier and mechanical properties, can be tailored by the processing parameters such as the temperature, time and pressure used in the process. The interface preferably has two functions: (i) a membrane function to control the exchange of fluid and species form one layer the other and (ii) a mechanical function, which consist of bonding of the layers and/or stiffening of the overall product. The multi-layered structure is used in systems were (i) specific properties are required in the different layers of the product and ( ii) the diffusion and transport between the two layers has to be controlled. Such systems are encountered in engineering devices and biomedical implants. In biomedical applications for instance, it could be used for the regeneration of tissues such as the osteochondral tissue.

The present invention relates to biocompatible, biodegradable and/or bioresorbable multi- layered cellular or porous composite structure also named foam, which integrates at least a functional interface, also named membrane, which may provide unique barrier and mechanical properties.

DETAILED DESCRIPTION OF THE INVENTION

The invention will be better understood below with a detailed description including non- limiting examples illustrated by the following figures.

As depicted in Figure 1, the composite is a multi-layered porous polymer matrix incorporating at least one interface. Cellular or porous structures exhibit open or closed pores, also named cells, separated by walls. Porosity is defined in terms of relative volume of pores and of pore size distributions. The foam density, defined as the relative mass of solid for a given volume, is related to the porosity. The porosity is given by 1 minus the ratio of foam density by solid density. Porosity can be closed and/or open when the pores are interconnected. The control of pore size is important for applications where liquids or biological media are injected or where growth of living cells occurs into the porous structure. The three-dimensional porous matrices, named porous layers, preferably have a plurality of interconnected pores (fiber meshes, sponges, foams, non woven fabrics).

The porosity, the material composition and the mechanical properties of the layers can be the same or different. It is preferred to have at least 70 % to 95 % of porosity. The material can be composed of a polymer, either as homogeneous or a combination of two or more polymers, as blend or copolymer, for example polylactide blended with PEG or copolymers of polylactide and PEG, or a composite of polymer and ceramic. For example, the devices produced for prototype (Figure 1) is composed of PLA/p-TCP composite (bottom layer) and PLA/PEG blends or copolymers (top layer). The layers are preferably cylindrical, conical or cubes or parallelepiped. The structure could contain pharmacologically or biologically active ingredients.

The membrane, also named functional interface in Figure 1, is located inbetween the porous layers. In a preferred embodiment the process for joining the two layers and create the membrane consists in (i) using a thermal source that heats the surface of at least one thermoplastic foam to the formation of a thin layer of molten polymers at its surface and then (ii) establishment of an intimate physical contact between the two foam surfaces to be joined, followed by macromolecular diffusion across the interface and by solidification of the polymers. The efficiency of these bonding phenomena is influenced by the cellular structure of the combined foams. The obtained material at the interface thus results from the interdiffusion of the polymer chains of the polymer of the assembled layers. Assembled materials should be compatible to promote molecular interdiffusion. The membrane thickness ranges preferably from 0.1 mm to 2 mm. An additional layer or thin porous film can be added inbetween two incompatible materials using the same processing route.

Created interfacial membranes can be impermeable and permeable membranes. Indeed the control of the processing parameters offers different porosity and density to the obtained interfaces. The membrane is considered impermeable to defined species (fluid, cells, vessels), when these species cannot pass form one layer to the other through the interface. The pore size can be tailored in a way to limit species of a defined size to go through the membrane while hindering species of larger size to pass. The porosity can be interconnected through the interface or closed. Examples are represented respectively on Figure 2 with types 1 and 2 of the novel cellular composites. Combinations of several layers and membranes types are possible (close or interconnected). Figure 3 gives an example of a multilayered porous structure composed of several layers of various porosity and thickness. The membrane density can be adjusted to tailor the permeability of the membranes to control the fluid flow through the interface. The porosity of the membrane is typically in a range between 0 % to 70 %. Furthermore, the membrane acts as barrier impermeable to agents that have a detrimental effect on the regeneration of the tissue to regenerate. The membrane, as shown on Figure 4, has a higher density than the foam layers offering higher mechanical properties at the interface. The membrane can be considered as well as a mechanical stiffener, that reinforces the overall mechanical performance of the structure. The density of the membrane can be tailored accordingly to adapt the stiffening of the structure to a given application.

An item according to the present invention may be seeded with suitable cells (differentiated or non-differentiated) prior to, during and immediately following the implantation. The micro structure can also be impregnated with a hydrogel and include a bioactive agent.

Membrane and structure fabrication

In one embodiment, the method to manufacture the functional membrane integrated in the multi-layered composites of the invention comprises several steps:

1) To provide the top and bottom porous layers composed of polymer and composite materials as described above. The layers of suitable shapes have at least one flat face of same form and size.

2) To provide a heat source and a clamping device as shown on Figure 5. The heat source is here based on convection and conduction mechanisms. The preferred examples are hot plate in convection and conduction or hot gas flow.

3) To heat the surface of at least one of the said end faces surface, using the mentioned heat source, to create a thin layer of molten polymer (Figure 5 and 6). The temperature of the heat source, the relative position of the surface to the heat source and the exposition time are critical processing parameters. 4) To dispose the two layers in a way that the surfaces face parallel in a mirroring position to each other, using a clamping device to control precisely the distance between the layers.

5) To displace the layers towards each other end face. The displacement and the time are controlled to allow bonding of the layers and generation of a functional interface.

The joining method to assemble the different layers does not involve the use of any solvents or chemical reactions. The bonding results from interdiffusion mechanisms and interpenetration of the polymers, which are heated at a temperature either above the melting temperature in the case of semi-cristalline thermoplastic or above the glass transition temperature for the amorphous polymers. The temperature at the surface of the porous layer Surface are preferably in the following range:

Amorphous thermoplastic: r_surf_ace = [T_g (glass transition temperature), T_g + 150 °C]

Semi-cristalline thermoplastic : r_surf_ace = [T_m (melting temperature), T_m + 60 °C]

Figure 7 provides an example of a processing window, which shows the domain of strong bonding without degradation of the material as a function of the exposition time and the heat source temperature for PLA based materials. The preferred heating time using a hot plate is below 10 seconds.

Another method to produce interfaces is the co-foaming of two or more polymer layers. In this case, a preform, composed of at least two layers of different material composition and/or thickness, is foamed in a mold by gas foaming. During this solvent free physical foaming process, the layers are melted and interdiffusion and interpenetration occur between the polymers of each layer. The porous structure is then obtained by expansion of the polymer and at the same time a membrane is generated at the interface of the porous layers. An example of a prototype, composed of case polylactide and a copolymer of polylactide and PEG layers, is given in Figure 8 showing a distinct interface inbetween the two porous layers of different morphologies. This method also does not involve any solvent. This method offers less flexibility for the selection of the porosity and final properties of the two layers while having the advantage of being a one step process.

During the interface formation by intimate contact and bonding of the layers, the size and the morphology of the pores at the interface are modified, with respect to the initial pore size and morphology of the pores in the layers. Within the interface, the pore size and the size of the interconnection between the pores are decreased and the density of the cellular structure is increased. The pore size may be reduced while maintaining the interconnectivity between the pores or while closing totally the interconnection between the pores. The reduction of pore size, interconnection size and the modification of pore morphology at the interface is generated by the pressure of contact, which results in mechanical stress on the pores at the interface, and by the temperature of the polymer, which allow plastic deformation of the pores. The permeability of the membrane, given by the final size of the pores at the interface and size of the interconnections between the pores, can be tailored by precise control of the contact pressure and polymer temperature during intimate contact. As schematized in Figure 6, the membrane can be semi-permeable or non-permeable. For example, to avoid vascular ingrowth through the membrane, the size of the interconnection of the interface should be preferably reduced to below 4 μιη. The reduction of pore size and the associated increase of density can also be realized to tailor the increase of stiffness of the cellular structure. MATERIAL SYSTEMS

Thermoplastic polymers in general can be used, such as polyethylene terephtalate (PET), polyethylene (PE), polyurethanes (PUR), etc. They may be reinforced with fillers, fibres or fabrics. The materials should be foamable and melt locally upon heat application.

Biocompatible and biodegradable polymers, fibres and particles already used in the biomedical field can be considered to prepare respectively biocompatible and biodegradable porous products to be used as scaffolds for example. Some examples of suitable polymers are cc-polyhydroxy acids, such as poly(glycolic acid) (PGA), poly(lactic acid) (PLA, L or D,L enantiomers), poly(e-caprolacton) (PCL), poly(trimethylene carbonate), poly(ethylene oxide) (PEO), Polyethylene glycol) (PEG), poly( -hydroxylbutyrate) (PHB), poly( -malic acid) (PMLA), poly( -hydroxyvalerate), poly( -hydroxypropionate) (PHPA), polyvinyl alcohol (PVA), poly hydroxyalkanoate (PHA), poly(p-dioxanone) (PDS), poly(ortho esters), poly(ester-urethane) (PEU), polypeptides, polysaccharides, elastin, fibrin, polyphosphazenes, as well as blends and copolymers of the above. Blends of the above with biocompatible plasticizer such as PEG, glycerol, citrate ester, oligomeric lactic acid (OLA) are also considered. During assembling, the foams must preferably be dried before processing, in order to prevent polymer hydrolysis. Layers of the proposed structure can be a thermoplastic composite based on the above- mentioned polymers combined with fillers or fibres. Some suitable examples are ceramic particles of calcium phosphates, such as hydroxyapatite (HAp), β-tricalcium phosphate (β- TCP), calcium carbonate (CC), calcium dihydrogenphosphate (CDHP), calcium hydrogenphosphate (CHP), or mixtures of above, and bioactive glasses, such as Bioglass^®, phosphate based glasses... Long and continuous fibres can be made of traditional materials such as glass, carbon, of resorbable glass, such as Bioglass^® and phosphate based glasses. Polymer and composite fibres are also used. The multi-layered foams obtained can be tailored in terms of open porosity. Obviously they can thus be infiltrated and filled with any media, liquid or gel bringing additional function to the structure. For example bioactive or rheoactive fluids are of interest for tissue engineering applications and damping materials.

Example I

An osteochondral graft consisting of a porous top and bottom layers, which are structurally integrated by an interface acting as a membrane.

The objective of this example is to illustrate how to obtain a multi-layered porous structure incorporating a membrane. This implant is for use in restoring damaged articular cartilage and also subchondral bone. Polymer foams of blends of PLLA with 20 wt PEG35000 (top layer) as well as a polymer- ceramic composite composed of PLLA filled with 5 wt β-tricalcium phosphate (β-TCP) microparticles (β-tri-Calcium phosphate, Fluka) (bottom layer) were obtained by C0₂ supercritical foaming. In this case, the porosity was 89 % and 78% for the top and bottom layer, respectively.

A set up was used to manipulate the two foams during the joining process. It allows one to maintain the surfaces of the foams parallel to the heat source during heating and parallel to each other when they are brought into intimate contact. The foams were held in a clamp maintained by two screws. The distance between the hot plate and the foam surface CIB on Figure 5) and the interpenetration length (L_; on Figure 5) could be precisely set using additional screws. The heating source used was a stainless steel plate heated at 420 °C. The foam surface was then placed at a defined distance (d =3.5 mm) from the plate and exposed to a temperature of 180 °C for 6 seconds. In a second step, the foam are brought into intimate contact maintaining a set L; of 0.5 to 2 mm until the solidification of the polymer. Parameters are indicated here for a poly(lactic acid) based system, but can obviously be adjusted for any type of thermoplastic based system.

By controlling the processing parameters a bi-layered scaffold for load-bearing surface of a joint, which integrates an interface is achieved as shown on Figure 1 and 2. First, the interface ensures a strong mechanical bond between the layers, second a membrane with a chosen porosity and barrier properties is fabricated. This membrane is expected to prevent the undesired ingrowth in the respective layers of the structure. The thickness of the barrier is preferably from about 20 μιη to about 1.5 mm and can be tailored by choosing the processing conditions. This barrier should limit the access of fibroblast, the vascular ingrowth and the haemoglobin from passing from the bottom part to the top part (i.e. from the bone part to cartilage part). The pore diameters of the membrane should be below 4 μιη to prevent blood to pass through the barrier. The barrier is also considered to provide significant stiffness to the overall scaffold. A supplementary layer, which would act as a gliding layer on top of the structure, could also be incorporated into the cartilage layer. This layer would be a porous structure such as a foam or an hydrogel.

The porous top layer corresponds to the cartilage layer and is intended to be placed accordingly when implanted. The height of this layer, which is defined as the distance between the interface and the top surface, varies from 3 to 10 mm, depending on the implantation site. The porous bottom layer corresponds to the subchondral bone and is intended to be placed accordingly when implanted. The height of this layer, which is defined as the distance between the interface and the bottom surface, varies from 3 to 20 mm, depending on the implantation site. The graft can be implanted into the defect site as it is or the surgeon may easily shape the scaffold using cutting tools to adapt it to perfectly fit to the defect. Example II

A bone graft that incorporates mechanical stiffeners

This scaffold for bone replacement in a load bearing area is composed of composite foams of PLLA reinforced with 5 wt β-tricalcium phosphate (β-TCP) microparticles (β-tri-Calcium phosphate, Fluka), or any other type of reinforcement (fillers, fibers), produced by C0₂ supercritical foaming with a porosity above 75 %. In this case, as seen on Figure 4, the scaffold incorporated several interfaces that are oriented in the axis of the load. Therefore, the scaffold mechanical properties in compression and shear are significantly improved by the interfaces that act as mechanical stringers. This stiffening mean offers the advantage to stiffen the scaffold while maintaining the interconnectivity of the pores in the overall scaffold or, if the application requires to guide the bone growth in a defined direction, the membrane could also be impermeable.

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Claims

1- A biocompatible multi-layered structure comprising a first foam layer showing a first degree of porosity and a second foam layer showing a second degree of porosity, both foam layers being attached to one another through an interface, wherein said interface is obtained from at least one of said foam layers and has a degree of porosity which is less than the degree of porosity of any of said foam layers.

2- A structure according to claim 1 wherein said interface is obtained from both said foam layers.

3- A structure according to claim 1 or 2 wherein said interface is obtained by heating the surface of at least one of said foam layers.

4- A structure according to claim 3 wherein said foam layers are made of thermoplastic or thermoplastic composite materials.

5- A structure according to claim 4 wherein said thermoplastic material is biocompatible and/or bioresorbable.

6- A structure according to anyone of the previous claims wherein said interface is a non- permeable membrane or a semi-permeable membrane with local values of porosity being between 0 and 70 %

7- A structure according to claim 6 wherein the said membrane is a functional membrane acting as a barrier and/or a mechanical stiffener.

8- A structure according to anyone of the previous claims wherein the membrane has a thickness comprised between 0.1 mm and 2 mm.

9- A structure according to anyone of the previous claim where the said foam layers and membrane are characterized by the fact that they are loaded with a pharmacologically or biologically active ingredients. 10- A structure according to anyone of the previous claim where the said porous layers are seeded with cells and/or impregnated with a hydrogel.

11- A structure according to anyone of the previous claims comprising more than two foam layers attached to one another through an interface.

12- A process for manufacturing a multi-layered structure as defined in anyone of the previous claims comprising the following steps:

a) the preparation of the foam layers

b) the local heating of at least one layer surface

c) the intimate physical contact between at least two layers with a controlled time, pressure and interface temperature

d) the cooling of the multi-layered structure

13- A process according to claim 12 that is a solvent free process.

14- A process according to claim 12 or 13 wherein local surface heating is obtained by convection and/or conduction from heating elements.

15- A process for manufacturing a multi-layered structure as defined in claims 1 to 11 comprising the following steps:

a) the stacking of polymer preforms in a mold

b) the co-foaming by a solvent-free gas foaming process

c) the in situ creation of the membrane

d) the cooling of the multi-layered structure