Patent Description:
The extended life expectancy and the raise of accidental trauma call for an increase of osteoarticular surgical procedures. Arthroplasty, the main clinical option to treat osteoarticular lesions, has limitations and drawbacks.

Regeneration of osteochondral defects represents a major challenge, especially considering the ageing of the population and the high impact on the public health system. The surgical procedures currently applied (bone graft, mosaicplasty, micro-fracture, articular prosthesis, therapeutic implant), are invasive and/or painful for the patient, with limited efficacy and side effects. Lesions of the femoral condyles are especially common, and can have serious consequences. A <NUM> study found that <NUM>% of patients undergoing arthroscopy showed osteochondral defects; in more than half of the cases, such a lesion was classified as grade <NUM> or higher, according to the International Cartilage Repair Society (ICRS) scale. Osteochondral defects do not heal properly and, even when treated (e.g. by Pridie's marrow stimulation or by mosaicplasty treatment) lead to osteoarthritis (OA) in <NUM>% of the cases. The unique properties of the cartilage (multilayered cell structure, different extracellular matrix composition and fibril orientation) make it difficult to repair. Surgical techniques like micro-fracture, mosaicplasty, osteoarticular transplantation or autologous chondrocytes implant may allow a partial functional recovery, but are mostly aimed to relieve the pain and prevent the lesion to spread (<NPL>)). All these techniques have a variable outcome (<NPL>)) and intrinsic limitations (<NPL>); <NPL>)) and none was shown to restore the hyaline articular surface (<NPL>)), justifying the search for alternative therapies to promote osteoarticular regeneration (OAR). Recently, membrane-based collagen material of mammalian origin (<NPL>); <NPL>); <NUM>. <NPL>); and <NPL>)) containing pre-cultured autologous chondrocytes, was used to fill articular focal lesions and promote cartilage regeneration. However, when performed on subchondral bone, they showed site morbidity and fibrocartilage formation (<NPL>)), leading to misfunctional repair. To overcome these limitations, mesenchymal stem cell (MSCs)-based therapies emerged, which employ autologous bone marrow derived MSCs to increase the efficiency of OAR (<NPL>); <NPL>); <NPL>); and <NPL>))). A combination of biomaterials, stem cells and active molecules are therefore needed to promote an effective tissue repair and to achieve a functional recovery of the articulation (<NPL>); and <NPL>)).

<CIT> discloses a biomaterial comprising a nanofibrous polymeric scaffold, said scaffold being coated with at least one layer pair consisting of a layer of polyanions and a layer of polycations, wherein said at least one layer pair incorporates a therapeutic molecule, said biomaterial also comprising living cells.

The aim of the present invention is thus to provide a new biomaterial able to address both subchondral bone and cartilage regeneration.

Another aim of the present invention is to provide a biomaterial useful for the prevention of osteoarthritis.

The present invention relates to the optimisation of the synthetic patches with different functions in an implant, a wound patch and protection covering. This innovative strategy offers major advances in the field of subchondral bone and cartilage regeneration, including multi-tissue differentiation (bone/cartilage) and local, precise stimulation by growth factors in adequate dosage (nanoscale).

The present invention relates to a biomaterial comprising:.

wherein the hydrogel (b) is included between the membrane wound patch (a) and the bone wound patch (c).

The present invention also relates to a biomaterial comprising:.

Preferably, the membrane wound patch is made of a nanofibrous polymeric scaffold, wherein said polymeric scaffold has a surface being optionally coated.

Preferably, the membrane wound patch is made of a nanofibrous polymeric scaffold, wherein said polymeric scaffold has a coated surface as explained above.

The scaffold of the patch (a) is made of a polymer. Preferably, the polymer is chosen from the group consisting of: poly(ε-caprolactone), collagen, fibrin, poly(lactic acid), poly(glycolic acid), poly(ethylene glycol)-terephtalate, poly(butylenes terephtalate), or co-polymers thereof, and mixtures thereof. The scaffold of the patch (a) can also be made of polymers such as hyaluronic acid, hydroxyapatite, chondroitin sulfate, chitosan, and mixtures thereof.

According to an embodiment, the nanofibrous polymeric scaffold of the membrane wound patch (a) is made of a polymer selected from the group consisting of: polyesters, polyamides, polyurethanes and polyureas, poly(amide-enamine)s, polyanhydrides, polymers produced from microbial, vegetal, marine or animal sources, and polymer blends thereof.

More preferably, the polymeric scaffold of the membrane wound patch (a) is made of poly(ε-caprolactone) or of collagen.

Most preferably, the nanofibrous polymeric scaffold of the membrane wound patch (a) is made of poly(ε-caprolactone), optionally mixed with chondroitin sulfate and hyaluronic acid.

According to the invention, the membrane wound patch comprises cartilage components, most preferably chondroitin sulfate and hyaluronic acid. Hyaluronic acid (HA) is a polysaccharide abundant in cartilaginous matrices, which constitutes an ideal chondrogenic microenvironment, ideal for cartilage regeneration. Chondroitin sulfate is an important structural component of cartilage and provides much of its resistance to compression.

According to an embodiment, the chondroitin sulfate and/or the hyaluronic acid are included in the polymeric scaffold. In such embodiment, the scaffold is not further coated.

According to an embodiment, the chondroitin sulfate and the hyaluronic acid are included in the coating of multilayered droplets, as polyanions.

According to a preferred embodiment, the membrane wound patch (a) is made of a poly(ε-caprolactone) scaffold including chondroitin sulfate/hyaluronic acid droplets.

According to a preferred embodiment, the poly(ε-caprolactone) is preferably electrospun.

In a specific embodiment, the scaffold of the patch (a) has a surface coated with an interrupted coating of multilayered droplets. These droplets may also be named "nanoreservoirs" or "nanocontainers".

More specifically, the scaffold of the patch (a) is coated, on a layer-by-layer basis, with layers that are alternatively negatively or positively charged.

This coating allows functionalizing the scaffold with a therapeutic molecule in such a way as to create nano-reservoirs of therapeutic molecules, as explained hereafter.

The term "multilayered droplet" refers to droplets or patches composed of at least one layer pair consisting of a layer of polyanions and a layer of polycations. Said droplets can present different shapes: circle shaped, oval-shaped or scale shaped. Preferably, said droplets have a size of <NUM> to <NUM>, more preferably <NUM> to <NUM>, even more preferably <NUM> to <NUM>.

According to the invention, the term "multilayered droplet coating" refers to a coating of droplets or patches disposed at the surface of the scaffold and obtained by layer-by-layer (LbL) deposition of oppositely charged molecules multilayered droplet.

The term "multilayered droplet coating" further refers to an interrupted coating of the scaffold, i.e. a coating that is not in the form of a continuous film along the surface of the biomaterial scaffold. The multilayer droplet coating may be characterized by its irregular shape and/or by the fact that it does not cover the totality of the surface of the scaffold, in such a way that at least a part of the surface of the scaffold is not coated. The multilayer droplet coating of the invention may be contrasted with a film coating having a smooth surface and covering the totality of the scaffold surface.

The building of the coating is based on the layer-by-layer (LbL) deposition of oppositely charged molecules. That is to say, the coating of the scaffold of the patch (a) is made in the same manner as is made a polyelectrolyte multilayered film. The bone wound patch (a) thus comprises polyelectrolyte multilayers, in the form of numerous multilayered droplets, on the surface of the scaffold.

More specifically, the nanofibrous scaffold according to the invention is coated, on a layer-by-layer basis, with layers that are alternatively negatively or positively charged. At least one of these layers incorporates and/or consists of the therapeutic molecule. These layers form "multilayered droplet" on the surface of the nanofibrous scaffold. This coating allows functionalizing the nanofibrous scaffold with a therapeutic molecule in such a way as to create nano-reservoirs of therapeutic molecules. The term "multilayered droplet" refers to droplets or patches composed of at least one-layer pair consisting of a layer of polyanions and a layer of polycations. Said droplets can present different shapes: circle shaped, oval-shaped or scale shaped. Preferably said droplets have a size of <NUM> to <NUM>, more preferably <NUM> to <NUM>, even more preferably <NUM> to <NUM>.

According to a preferred embodiment, the polycations are chosen from the group consisting of: poly(lysine) polypeptides (PLL), covalently-coupled cyclodextrin-poly(lysine) (PLL-CDs), poly(arginine) polypeptides, poly(histidine) polypeptides, poly(ornithine) polypeptides, Dendri-Graft Poly-lysines (e.g. Dendri-Graft Poly-L-lysines), chitosan, and mixtures thereof.

More preferably, the polycation is chitosan.

According to a preferred embodiment, the polyanions may also include further polyanions in addition to the chondroitin sulfate and hyaluronic acid. Such further polyanions are chosen from the group consisting of: poly(glutamic acid) polypeptides (PGA), poly(aspartic acid) polypeptides, and mixtures thereof.

According to an embodiment, the coating of multilayered droplets is interrupted, said multilayered droplets being droplets composed of at least one layer pair consisting of a layer of polyanions including at least chondroitin sulfate and/or hyaluronic acid, and a layer of polycations.

The polyelectrolyte multilayers that coat the nanofibrous scaffold of the patch (a) are composed of at least one layer pair consisting of a layer of polyanions including at least chondroitin sulfate and hyaluronic acid and of a layer of polycations. They may for example comprise <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or more layer pairs. Preferably, it comprises from <NUM> to <NUM> layer pairs.

The preparation of such coating is further explained below.

According to an embodiment, the polymeric scaffold of the patch (a) is coated with a continuous coating comprising more than <NUM> layer pairs, each layer pair consisting of a layer of polyanions including at least chondroitin sulfate and hyaluronic acid, and a layer of polycations.

Preferably, according to such embodiment, the nanofibrous scaffolds are coated with fifteen to thirty layer pairs each consisting of a layer of polyanions (including hyaluronic acid and/or chondroitin sulfate, which are negatively charged), and a layer of polycations (namely DGLG5, which is positively charged).

The advantage of the multilayered droplet coating including hyaluronic acid or chondroitin sulfate compared with a multilayered droplet coating including BMP2 is the modification of the mechanical properties of the nanofibrous scafold.

Consequently, the two cartilage components will give hydrophilic and viscous cartilage-like composition helping to reduce articular friction therapeutic molecules improve the mechanical resistance of scaffold and enhance the tissue regeneration through their therapeutic effect.

A preferred embodiment, the nanofibrous scaffold is preferably a coated film. The coating according to the invention is preferably, regularly spread over the nanofiber surface. Preferably the continuous coating comprising more than <NUM> layer pairs, each layer pair consisting of a layer of polyanions and a layer of polycations. They may for example comprise <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or more layer pairs. Preferably, it comprises from <NUM> to <NUM> layer pairs.

According to the invention, the term coated film refers to a coating disposed at the surface of the nanofiber and obtained by layer-by-layer (LbL) deposition of oppositely charged molecules. Due to the repartition of the surface charges of the polymer constituting the nanofiber, the first layer of polyanions or polycations form small droplets or patches adsorbed along the surface of the nanofibers. At each step of the polyanions or polycations polymer application, each droplet is covered by a new layer of polyanions or polycations polymer. The coating process cab be stopped when a film coating is observed. When the film coating is obtained, the multilayered droplet cannot be obtained any more along the surface of the coated nanofiber.

The term "continuous coating film" further refers to a continued coating of the nanofibers, i.e. a coating in the form of a continuous film along the surface of the nanofibers. The continuous coating film may be characterized by its regular shape and/or by the fact that it cover the totality of the surface of the nanofiber, in such a way that at least a part of the surface of the nanofiber is not coated. The continuous coating have a smooth surface and covering the totality of the nanofiber surface (see <FIG>).

According to the invention, this preferred continuous coating comprises chondroitin sulfate and/or hyaluronic acid as polyanions.

The coated film provides advantageous characteristics to the nanofiber for its use as membrane wound patch. The first advantage of the film coating compared with the multilayered droplet coating is its mechanical property to secure and protect the other parts of implant. The other advantage is the combination of the slow degradability and the strong quantity of therapeutic molecule that can be applied on the nanofibrous, chondroitin sulfate and/or hyaluronic acid, which decrease the risk of inflammation and enhance the regeneration of for a long time.

In a preferred embodiment, the PCL is preferably a biphasic electrospun PCL membrane. Preferably, according to such embodiment, this membrane can be composed of random fibres covered with a layer of aligned fibres. This biphasic membrane can for example be obtained as described in the two first paragraphs of Example <NUM> with nanoreservoirs of CS/HA.

Preferably, the hydrogel (b) comprises hyaluronic acid and/or alginate.

As mentioned above, the hydrogel comprises living cells being autologous or allogenic bone marrow-derived mesenchymal stem cells. Preferably, the hydrogel comprises autologous bone marrow-derived mesenchymal stem cells. Said living cells are preferably obtained by induced pluripotent stem cells (iPSCs) technology.

In a specific embodiment, said living cells are comprised within a hydrogel (e.g. an alginate hydrogel or a collagen hydrogel) that is deposited on the bone wound patch (c) scaffold.

Hydrogels are well-known to the skilled in the art. An alginate hydrogel may for example be a mixture of alginate and hyaluronic acid (e.g. a alginate:hyaluronic acid solution (<NUM>:<NUM>), which may be prepared in a <NUM> NaCl solution at pH <NUM>).

The scaffold of the patch (c) is made of a polymer.

Preferably, the polymer is chosen from the group consisting of: poly(ε-caprolactone), collagen, fibrin, poly(lactic acid), poly(glycolic acid), poly(ethylene glycol)-terephtalate, poly(butylenes terephtalate), or co-polymers thereof, and mixtures thereof.

According to an embodiment, the nanofibrous polymeric scaffold of the bone wound patch (c) is made of a polymer selected from the group consisting of: polyesters, polyamides, polyurethanes and polyureas, poly(amide-enamine)s, polyanhydrides, polymers produced from microbial, vegetal, marine or animal sources, and polymer blends thereof.

More preferably, the polymeric scaffold of the bone wound patch (c) is made of poly(ε-caprolactone) or of collagen.

As mentioned above, the scaffold of the patch (c) has a surface coated with an interrupted coating of multilayered droplets. These droplets may also be named "nanoreservoirs" or "nanocontainers" and are as defined above for the patch (a).

According to some embodiments, the scaffold is multilayered droplet coated.

The coating of the patch (c) is preferably irregularly spread over the scaffold surface.

More specifically, the scaffold of the patch (c) is coated, on a layer-by-layer basis, with layers that are alternatively negatively or positively charged.

The term "multilayered droplet coating" further refers to an interrupted coating of the scaffold of (c), i.e. a coating that is not in the form of a continuous film along the surface of the polymeric scaffold of (c). The multilayer droplet coating may be characterized by its irregular shape and/or by the fact that it does not cover the totality of the surface of the scaffold, in such a way that at least a part of the surface of the scaffold is not coated. Such multilayer droplet coating may be contrasted with a film coating having a smooth surface and covering the totality of the scaffold surface.

The building of the coating is based on the layer-by-layer (LbL) deposition of oppositely charged molecules. That is to say, the coating of the scaffold of the patch (c) is made in the same manner as is made a polyelectrolyte multilayered film. The bone wound patch (c) thus comprises polyelectrolyte multilayers, in the form of numerous multilayered droplets, on the surface of the scaffold.

In contrast to a film coating that covers the whole scaffold surface, the multilayered droplet coating preferably only partially covers the scaffold surface. The coating according to the invention is applied layer by layer (LbL), the excess amount of polyanions or polycations is removed at each step with rinsing steps between consecutive adsorption steps. Due to the repartition of the surface charges, the first layer of polyanions or polycations form small droplets or patches adsorbed along the surface of the scaffold. At each step of the polyanions or polycations application, each droplet is covered by a new layer of polyanions or polycations. The coating process is stopped when the multilayered droplet coating is observed and before a film coating. The multilayered droplet coating provides advantageous characteristics to the scaffold, which are not observed with a film coating. When the film coating is obtained, the multilayered droplets cannot be obtained any more along the surface of the coated scaffold.

The first advantage of the multilayered droplet coating compared with the film coating or the uncoated scaffold is its irregular surface. This irregular shape improves the adherence of cells to the scaffold. Moreover, this irregular shape provides an increase of the surface of contact between the coating and cells, optimizing the exchanges between the coating and cells. Consequently, a small concentration of therapeutic molecule (if present) is needed for observing a better stimulation of cell growth.

In addition, the coating of the invention uses fewer polyanions and polycations layers than the film coating. A reduced number of layers are thus needed to obtain the multilayered droplet coating.

As further used herein, the term "polyelectrolyte multilayers" notably encompasses the multilayered droplets that coat the scaffold of the patch (c) according to the invention.

In the frame of the present specification, the term "polyelectrolyte" designates compounds that bear several electrolyte groups, in particular polymers whose repeating units carry electrolyte groups. The groups will dissociate in aqueous solutions, giving rise to polyanions or polycations, as the case may be, and making the polymers charged.

The polyelectrolyte multilayers that coat the nanofibrous scaffold of the patch (c) are composed of at least one layer pair consisting of a layer of polyanions and of a layer of polycations. They may for example comprise <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or more layer pairs. Preferably, it comprises from <NUM> to <NUM> layer pairs.

Polyelectrolyte multilayers, and in particular multilayered droplet as described herein, can easily be obtained by the alternate dipping of the scaffold of the patch (c) in polyanion and polycation solutions.

As apparent to the skilled in the art, the only requirement for the choice of the polyanions and polycations is the charge of the molecule, i.e., the polyanion shall be negatively charged and the polycation shall be positively charged. The polyanions and polycations according to the invention may correspond to any type of molecule, such as e.g. a polypeptide (optionally chemically modified) or a polysaccharide (including cyclodextrins, chitosan, etc.).

According to a preferred embodiment, the polyanions are chosen from the group consisting of: poly(glutamic acid) polypeptides (PGA), poly(aspartic acid) polypeptides, and mixtures thereof.

As mentioned above, the bone wound patch (c) comprises a growth factor.

Preferably, the growth factor is selected from the group consisting of: a vascular endothelial growth factor (VEGF), a bone morphogenetic protein (BMP), such as BMP2, a transforming growth factor (TGF), a fibroblast growth factor (FGF), a nucleic acid coding therefor, and mixtures thereof.

More preferably, the bone wound patch (c) is made of a poly(ε-caprolactone) scaffold including chitosane/BMP2 droplets.

Therapeutic molecules such as growth factors can be incorporated into polyelectrolyte multilayers, as described, e.g., in <CIT>, <CIT>, <NPL>), <NPL>) and <NPL>).

When the biomaterial according to the invention is used for bone and/or cartilage regeneration, said growth factor is most preferably selected from the group consisting of bone morphogenetic protein <NUM> (BMP2), bone morphogenetic protein <NUM> (BMP4), bone morphogenetic protein <NUM> (BMP7), fibroblast growth factor <NUM> (FGF1), fibroblast growth factor <NUM> (FGF2), fibroblast growth factor <NUM> (FGF4), fibroblast growth factor <NUM> (FGF8), fibroblast growth factor <NUM> (FGF9) and fibroblast growth factor <NUM> (FGF18).

In a specific embodiment, the polyelectrolyte multilayers comprise or consist of at least <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or more layer pairs, each layer pair consisting of:.

The present invention also relates to a method for preparing the bone wound patch (c) as defined above, said method comprising a step of coating the scaffold with at least one layer pair consisting of a layer of polyanions and a layer of polycations.

Preferably, the above-mentioned step of coating with at least one layer pair comprises the following steps:.

At step (i) and (iii), the solution comprising the polycations or polyanions may for example comprise a concentration of polycations or polyanions within a range of about <NUM> to about <NUM>, preferably of about <NUM> to about <NUM>. Said solution may for example comprise or consist of, in addition to the polyanions or polycations, <NUM> <NUM>-(N-morpholino)ethanesulfonic acid (MES) and <NUM> NaCl. The pH of the solution is preferably neutral (e.g. a pH of <NUM>).

At step (ii) and (iv), the scaffolds may for example be rinsed with a solution having a neutral pH (e.g. a pH of <NUM>). Said solution may for example comprise or consist of <NUM> MES and <NUM> NaCl.

Step (v) may be repeated any number of times, depending on the number of layer pairs that should coat the scaffold.

Step (vi) may for example be carried out by exposure to ultraviolet light (for example at <NUM>, <NUM> W, at an illumination distance of <NUM>, for about <NUM> to about <NUM> hour, preferably for about <NUM>).

Before use, the bone wound patch (c) according to the invention may be equilibrated (e.g. by bringing it in contact with serum-free medium).

As immediately apparent to the skilled in the art, the steps in which the nanofibrous scaffold is immersed in a solution comprising polycations or polyanions may be replaced with steps wherein said solution is sprayed onto the scaffold.

The hydrogel (b) as defined above may be prepared by a method comprising the following steps:.

The present invention also relates to a method for the treatment of cartilage lesion using the biomaterial as defined above, said method comprising a step of applying the bone wound patch (c), a step of applying the hydrogel (b) and applying the membrane wound patch (a).

The present invention also relates to a biomaterial as defined above, for use in bone and/or cartilage regeneration.

The present invention also relates to a biomaterial as defined above, for use in the treatment of a bone and/or cartilage defect.

The present invention also relates to a kit for use for the treatment and/or the prevention of osteoarthritis, comprising:.

as a combined preparation for simultaneous, separate or sequential use in the treatment and/or the prevention of osteoarthritis.

The present invention also concerns ARTiCAR, an innovative implant for the treatment of osteoarticular lesions that combines two advanced therapy medicinal products for addressing both subchondral bone and cartilage regeneration.

The present invention is thus based on ARTiCAR (ARTicular CArtilage and subchondRal bone implant) combined Advanced Therapy Medicinal Products (ATMPs) for personalized OAR (<FIG>). The implant is made of i) a nanofibrous FDA-approved resorbable polymeric (Poly-ε-caprolactone: PCL) wound dressing, nano-functionalized with Bone morphogenetic factor <NUM> (BMP2)-nanoreservoirs for cell-contact dependent local delivery of therapeutics, and ii) autologous bone marrow-derived MSCs, encapsulated into a hyaluronic acid/alginate-based hydrogel (<FIG>). The nanoreservoirs technology enabled to reduce the dose of BMP2 to physiological levels, making it locally and sustainably available, and reducing the adverse effects of its massive release, e.g. from the soaked collagen sponges currently used in the clinic.

The present invention thus also concerns a biomaterial comprising:.

According to an embodiment, this biomaterial comprises:.

said hydrogel (b) and said bone wound patch (c) being as defined above.

Preferably, the nanofibrous scaffold is made of poly(ε-caprolactone) or of collagen.

Preferably, the hydrogel is a hyaluronic acid/alginate-based hydrogel.

According to an embodiment, this biomaterial further comprises a therapeutic molecule within at least one multilayered droplet or forming at least one multilayered droplet when said therapeutic molecule is charged. Preferably, the therapeutic molecule is a growth factor selected from the group consisting of: a vascular endothelial growth factor (VEGF), a bone morphogenetic protein (BMP), a transforming growth factor (TGF), a fibroblast growth factor (FGF), a nucleic acid coding therefor, and mixtures thereof.

The present invention also concerns a method for the treatment of cartilage lesion using this biomaterial, said method comprising a step of nanofibrous scaffold application and a step of hydrogel application.

ARTiCAR consists of a polymeric nanofibrous bone wound dressing nano-functionalized with a growth factor to promote subchondral bone regeneration, and bone marrow mesenchymal stem cells embedded into hydrogel, for cartilage regeneration. In this work, the ARTiCAR was tested for i) the feasibility in treating osteochondral defects in a large animal model, ii) the possibility to monitor healing non-invasively and iii) the overall safety in two animal models under GLP preclinical standards. The data indicate the preclinical safety of ARTiCAR following international regulatory guidelines, which could undergo phase I clinical trial.

The safety of the ARTiCAR combined ATMPs was tested in promoting OAR in two different animal models. The results of the feasibility, toxicity and biodistribution tests, run accordingly to the international regulatory guidelines for cell therapies and medical devices and Good Laboratory Practice (GLP), proved the biosafety of the ARTiCAR combined ATMPs, which can therefore be used for phase I clinical trials as a ready-to-use, flexible implant to address both cartilage and subchondral bone regeneration.

Poly(ε-caprolactone) (PCL), analytical grade, was purchased from Sigma Aldrich. PCL was dissolved in a mixture of dichloromethane/dimethylformamide (DCM/DMF <NUM>/<NUM> vol/vol) at <NUM> % wt/vol and was stirred overnight before use. The Dendri Graft Poly-L-Lysines (DGLs) were purchased from COLCOM (Montpellier, France). In this study, the fifth-generation DGLG5 has been used. Human recombinant BMP2 was purchased from PeproTech. Sodium alginate medium viscosity was from Sigma and hyaluronic acid (M. <NUM>) from Lifecore. Rat-tail type I collagen was purchased from Institut de Biotechnologies Jacques Boy. Poly(L-lysine) (PLL) was purchased from Sigma and chitosan (CHI), Protasan up CL <NUM>, was from FMC Biopolymer (Norway). Human recombinant BMP-<NUM> was purchased from PeproTech.

A homemade standard electrospinning set-up was used to fabricate the PCL scaffolds. The PCL solution was poured into a <NUM> syringe and ejected through a needle with a diameter of <NUM> at a flow rate of <NUM>/h, thanks to a programmable pump (Harvard Apparatus). A high-voltage power supply (SPELLMAN, SL30P10) was used to set <NUM> kV at the needle. Aluminum foils (20x20 cm2), connected to the ground at a distance from the needle of <NUM>, were used to collect the electrospun PCL scaffold. The collected scaffold comprise uniform randomly oriented fibres (Li et al, <FIG>, <NPL>).

A second method comprises the use of polycaprolactone (PCL) solution (<NUM>% w/w), loaded into a <NUM> plastic syringe with an <NUM> needle and fed at a constant rate of <NUM>/h using a syringe pump. A positive voltage of 10kV was applied to the needle using a high-voltage power supply. The distance between the collector and the needle tip was set at <NUM>. A rotating disk was used as the collector of electrospun fibrous scaffolds. A high rotating speed (<NUM><NUM> to <NUM><NUM> rpm) was used for fabricating scaffolds composed of aligned fibres, while a low rotating speed (<NUM> - <NUM> rpm) was used for collecting scaffolds with randomly oriented fibres. The scaffolds were vacuum dried overnight prior to subsequent experiments.

For all biological activity experiments, polyelectrolyte multilayers were prepared on Electrospun PCL membrane. Multilayers constituted by (DGLG5-BMP2)n or (PLL-BMP2)n or (CHI-BMP2)n were built by alternating immersion of the surfaces during <NUM> in the respective solutions (<NUM>µl) at the respective concentrations of <NUM> for DGLG5 or PLL or CHI and <NUM> of BMP2 in presence of <NUM> MES and <NUM> NaCl at pH=<NUM>. After each deposition step the membranes were rinsed during <NUM> with <NUM> MES and <NUM> NaCl at pH=<NUM>. All the membranes were sterilized for <NUM> by exposure to ultraviolet (UV) light (<NUM>, <NUM> W, illumination distance <NUM>). Before use, all membranes were equilibrated in contact with <NUM> of serum-free medium (see Cell culture).

In addition, polyelectrolyte multilayers on a Bio-Gide® resorbable collagen membrane (Geistlich Pharma AG, Germany), instead of an Electrospun PCL membrane, were also built.

For all biological activity experiments, polyelectrolyte multilayers were prepared on Electrospun PCL membrane. Multilayers constituted by (DGLG5-CS/HA)n or (PLL-CS/HA)n or (CHI-CS/HA)n were built by alternating immersion of the surfaces during <NUM> in the respective solutions (<NUM>µl) at the respective concentrations of <NUM> for DGLG5 or PLL or CHI and <NUM> of a mix of Chondroitin sulfate and hyaluronic acid in presence of <NUM> MES and <NUM> NaCl at pH=<NUM>. After each deposition step the membranes were rinsed during <NUM> with <NUM> MES and <NUM> NaCl at pH=<NUM>. All the membranes were sterilized for <NUM> by exposure to ultraviolet (UV) light (<NUM>, <NUM> W, illumination distance <NUM>). Before use, all membranes were equilibrated in contact with <NUM> of serum-free medium (see Cell culture).

For all biological activity experiments, polyelectrolyte multilayers were prepared on Electrospun PCL membrane. Multilayers constituted by (DGLG5-CS/HA)n or (PLL-CS/HA)n or (CHI-CS/HA)n were built by alternating immersion of the surfaces during <NUM> in the respective solutions (<NUM>µl) at the respective concentrations of <NUM> for DGLG5 or PLL or CHI and <NUM> of of a mix of Chondroitin sulfate and hyaluronic acid in presence of <NUM> MES and <NUM> NaCl at pH=<NUM>. After each deposition step the membranes were rinsed during <NUM> with <NUM> MES and <NUM> NaCl at pH=<NUM>.

The deposition steps were made until the fiber surface was fully covered by a uniform polyelectrolyte coating, without any multilayered droplets. At least <NUM> bilayers were deposited on fiber with at least <NUM> deposition steps.

All the membranes were sterilized for <NUM> by exposure to ultraviolet (UV) light (<NUM>, <NUM> W, illumination distance <NUM>). Before use, all membranes were equilibrated in contact with <NUM> of serum-free medium (see Cell culture).

Human primary osteoblasts (HOB) were obtain from Cell Applications and cultured in Dulbecco's modified Eagle's medium (D-MEM®) containing <NUM> U/mL penicillin, 50µg/mL streptomycin, <NUM>. 5µg/mL Amphotericin B and <NUM>% FBS (Life Technologies, Paisley, UK). The cultures were incubated at <NUM> in a humidified atmosphere of <NUM>% CO2. When the cells reached sub-confluence, they were harvested with trypsin and sub-cultured.

The MSCs harvested from iliac crest were isolated according to their adherence to cell culture plastic. Bone marrow aspirates were first washed by addition of an equal volume of phosphate buffer saline (PBS; Sigma-Aldrich, France) and centrifuged at <NUM>×g for <NUM>. The cell pellets were suspended in Dulbecco's Modified Eagle Medium (DMEM; Lonza, Germany) containing <NUM>% heat-inactivated fetal bovine serum (Gibco, Thermo Fisher Scientific, France), <NUM> U/mL of penicillin (Lonza, Germany), <NUM>µg/mL of streptomycin (Lonza, Germany), <NUM>µg/mL Fungizone (Lonza, Germany), and seeded in a T75 culture flasks, under standard cell culture conditions. The following day, medium was discarded and attached cells were gently washed up several times with PBS to remove non-adherent cells. Flasks were then incubated for several days in DMEM, replaced every <NUM> to promote emergence of colonies from adherent cells. When cells finally reached sub-confluence, they were sub-cultured until passage <NUM>, when they were expanded for stemness characterization.

<NUM>×<NUM><NUM> human osteoblasts were seeded and incubated for <NUM> prior to gel preparation. For the collagen lattices preparation, <NUM> of Rat Tail Type-I Collagen (Institut de Biotechnologies Jacques Boy) were mixed with <NUM> of medium containing <NUM>% FBS, <NUM> of a <NUM> NaOH solution and <NUM> of cell suspension at 2x105 cells/ml. <NUM> of the cells suspension: collagen preparation were poured on the top of the electrospinned membrane and allow to polymerize by incubating it at <NUM> for <NUM>. After polymerization, <NUM> of a human chondrocyte suspension (1x105 cells/ml) in an alginate hyaluronic acid solution (<NUM>:<NUM>) prepared in <NUM> NaCl, pH <NUM> were poured on the top of the collagen lattice in order to obtain the <NUM>-layered construct. <NUM> or <NUM> cylinders were cute using an sterile biopsy punch and incubated o/n at <NUM> in a humidified atmosphere of <NUM>% CO<NUM> prior to in vivo experiments.

Cell viability was determined by trypan blue exclusion. AlamarBlue® (Serotec) was used to assess cellular proliferation. The Alamar Blue test is a non-toxic, watersoluble, colorimetric redox indicator that changes color in response to cell metabolism. In this study, <NUM>×<NUM><NUM> human osteoblasts were seeded on the top of LbL-coated <NUM>-diameter membranes (n=<NUM>) placed on <NUM>-well plates. After <NUM> days of culture, cells were incubated in <NUM>% AlamarBlue/DMEM solution in a humidified atmosphere at <NUM> and <NUM>% CO2. After <NUM> hours, <NUM> of incubation media was transferred to <NUM>-well plates and measured at <NUM> and <NUM> in order to determine the percentage of AlamarBlue reduction.

All the experiments in this study were planned and performed according to the international regulatory guidelines for cell therapies and medical devices. Good Laboratory Practice and Standard Operating Procedures (SOPs) for all protocols were used. In vitro cytotoxicity assay was done according to ISO <NUM>-<NUM> (<NUM> and <NUM>) guidelines. Assessment of the OAR was done in accordance to the ICRS II score system.

The nanofibrous component of ARTiCAR was obtained via electrospinning of PCL, as previously described (<NPL>)). Briefly, PCL (PURASORB®, PURAC, Corbion, Amsterdam, Netherlands) was dissolved in a <NUM>% (wt/vol) dimethylformamide/dichloromethane solution (<NUM>/<NUM>, v/v) and delivered at a constant rate of <NUM>/h to the EC-DIG electrospinning device (IME Technologies, Eindhoven, Netherlands), set to at high voltage (<NUM>+/-<NUM> kV). Following electrospinning, PCL membranes were kept in a desiccator at <NUM>, to remove residual solvents, and sterilised by gamma irradiations (<NUM> kGy). Membranes were then dipped alternately in <NUM>µg/ml BMP2 solution (rh-BMP2, Inductos, Medtronic, France) in <NUM> <NUM>-Morpholinoethanesulfonic acid (Sigma-Aldrich, Saint-Quentin Fallavier, France), <NUM> Sodium Chloride (Sigma-Aldrich), pH <NUM> (MES buffer) and <NUM>/ml Chitosan (Protasan UP CL <NUM>, Novamatrix, Sandvika, Norway), for <NUM> times. Each bath was followed by <NUM> washes in MES buffer.

Twelve mg/ml sodium alginate (Sigma-Aldrich) and <NUM>/ml hyaluronic acid (Lifecore Biomedical, Chaska, USA) were dissolved in <NUM>/ml Sodium Chloride (Sigma-Aldrich). Prior to implant, the hydrogel (b) was mixed with either human or sheep MSCs. After the MSC/hydrogel compartment was applied to fill the defect, gelation was achieved using <NUM> calcium chloride (Sigma-Aldrich).

MRC5 cells were plated into <NUM>-well plates. The NanoM1-BMP2 wound dressing was tested side by side with polyurethane film containing <NUM>% zinc diethyldithiocarbamate (known for inducing cytotoxic effects; Hatano Research Institute/Food and Drug Safety Center, Japan) and high density polyethylene film (negative control; Hatano Research Institute). To assess cytotoxicity, pieces of different size (<NUM><NUM>, <NUM><NUM>, <NUM><NUM>, <NUM><NUM>, <NUM><NUM>) were placed in contact to the cultured cells, when <NUM>-<NUM>% confluence was reached. Cells were cultured in the presence of the membranes for <NUM> days before being examined microscopically for changes in the general morphology, presence of vacuolization, detachment, lysis and membrane integrity, following the criteria for the qualitative evaluation of cytotoxicity according to ISO <NUM> guidelines, part <NUM> (<NUM>) and part <NUM> (<NUM>): Class <NUM>, no reactivity (no effects around or below sample); Class <NUM>, slight reactivity (few malformed or degenerated cells); Class <NUM>, mild reactivity (small area of malformed or degenerated cells below the sample); Class <NUM>, moderate reactivity (malformed or degenerated cells in an area larger than the size of the sample but ≤ 1cm2); Class <NUM>, severe reactivity (malformed or degenerated cells in an area larger than the size of the sample but > 1cm2). A grade higher than <NUM> was considered as cytotoxic.

As a quantitative measure of cytotoxicity, cell viability was evaluated. At day <NUM>, membranes were discarded, cells were washed twice with PBS, fed with <NUM> culture medium and <NUM>µl/well of Cell Viability Reagent WST-<NUM> (Lonza) was added to each well, according to internal SOPs. The cells were incubated for <NUM> at <NUM> in <NUM>% CO<NUM>, and <NUM>µl of supernatant were transferred into a <NUM>-well plate. Absorbance was measured at <NUM> and <NUM> in a Multiskan EX device (Thermo Fisher Scientific, Graffenstaden, France). Data analysis was performed with Ascent <NUM> (Thermo Fisher Scientific). Results were expressed as percentage of viable cells in respect to a blank control. A decrease of <NUM>% viability was considered as cytotoxic.

Animal experiments were performed according to the ethical guidelines for animal experiments. The protocols used, included in the project "Toxicologie Réglementaire", was authorized by the "Ministère de I'Enseignement supérieur et de la Recherche" No. <NUM>. Seven-weeks old rats were maintained for at least <NUM> days in Specific Pathogen Free rooms (authorized by the French Ministries of Agriculture and Research; agreement No. A35 <NUM>-<NUM>) before the beginning of the study, according to internal SOPs, under controlled conditions of temperature (<NUM> ± <NUM>), humidity (<NUM> ± <NUM>%), photoperiod (<NUM> light/<NUM> dark) and air exchange, according to internal SOPs. Animals were housed in standard-size polycarbonate cages (with filter lid), and bedding was replaced twice a week.

Evaluation of acute toxicity in vivo was achieved via intra-articular implant of ARTiCAR in a model of induced osteochondral defect in RH-Foxn1 rnu/rnu nude rats (Harlan, Gannat, France). Briefly, sterile NanoM1-BMP2 were rinsed in sterile PBS and cut into quarters (<NUM><NUM>) before implant. Subconfluent human bone marrow MSCs (Promocell, Heidelberg, Germany) were washed and resuspended in hyaluronic acid/alginate mixture to a concentration of <NUM> × <NUM> cells/ml, as previously published (<NPL>); and <NPL>)). Prior to implant, rats were anesthetized with intraperitoneal injection of a solution of <NUM>/kg ketamine and <NUM>/kg xylazine. After shaving and disinfection of right hind leg, round <NUM> osteochondral defects were induced with a short drill in the patellar groove of the femur, in the midline of the femoral trochlea, until bleeding of the subchondral bone (approx. The NanoM1-BMP2 membrane was placed at the bottom of the defect, which was in turn filled with hMSCs/ hydrogel mix and gelled via drop-wise addition of <NUM> calcium chloride (Sigma-Aldrich), over <NUM> minutes. These rats constituted experimental group <NUM> (ARTiCAR; n = <NUM> rats; <NUM> males and <NUM> females; <NUM>µl of hydrogel containing <NUM>,<NUM> ± <NUM>% cells). Other rats were subject to the same procedure, but implanted with hydrogel only (as vehicle) and constituted group <NUM> (n = <NUM> rats; <NUM> males and <NUM> females; <NUM>µl of hydrogel). After gelation, the articulation capsule was closed, muscle and skin were sutured and the wound was thoroughly disinfected with povidone-iodine solution. After surgery, rats were kept under observation for post-anaesthesia recovery. After recovery, <NUM>-<NUM>/ kg buprenorphine was administered by subcutaneous injection. Animals were allowed unrestricted movement for the duration of the study (<NUM> days). Rats were monitored daily for wound healing, leg mobility, morbidity, mortality and evident sign of toxicity.

At day <NUM> post implant, <NUM> male and <NUM> female fasted rats/group (n = <NUM>) were anesthetized with excess isoflurane and ventricular blood was collected either in EDTA-containing tubes or in heparin-containing tubes, for haematological or biochemical analysis, respectively. Between day <NUM> and <NUM>, the remaining rats were observed and monitored twice a week for any loss of weight. Haematocrit, haemoglobin concentration, erythrocyte count, leukocyte counts, mean corpuscular volume and platelet count were determined in the blood samples on the day of collection by impedance variation and photometry (MINDRAY BC <NUM> haematology analyser, <NUM>, France). For biochemistry evaluation, plasma samples were prepared according to internal SOPs. Sodium, potassium, chloride, calcium, inorganic phosphate, glucose, urea, creatinine, total bilirubin, total cholesterol, triglycerides, aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), total proteins, albumin, and albumin/ globulin ratio were quantified (Cobas Mira biochemistry analyser, <NUM>, France).

Histopathology analysis was conducted on the fasted animals used for blood test. Briefly, a macroscopic autopsy was performed on freshly euthanized rats. Organs (treated knee, spleen, mesenteric lymph nodes, liver, lungs with bronchi and bronchiole, kidneys and heart) were macroscopically observed, explanted and collected. The right hind paw was sectioned at the epiphyses of both femur and tibia to recover knee joints subject to implant. Spleen, liver, kidneys and heart were weighed and preserved with the other organs at room temperature in <NUM>% formalin (Sigma-Aldrich) until histological analyses. Organs were fixed in <NUM>% paraformaldehyde, dehydrated, embedded in paraffin, sectioned and examined for histopathology.

Ninety days post implant, <NUM> male and <NUM> female rats/group (n = <NUM>) were euthanized by exsanguination under anaesthesia. After a macroscopic autopsy organs (ovaries with oviducts, testes, brain, treated knee, spleen, liver, kidneys, lungs, bone marrow, heart and the skin covering the treated knee joint) were weighed and collected for DNA extraction using the NucleoBond AXG100 kit (Macherey Nagel, Hoerdt, France), following manufacturer's instructions. Briefly, all tissue samples except knee joints were homogenized in M-tubes (Miltenyi Biotec, Paris, France) containing buffer G2 on a GentleMACS Dissociator (Miltenyi Biotec, Paris, France). Knee joints were homogenized using Ultra-Turrax® dissociator instrument in buffer G2. Following extraction, the DNA pellet was dissolved in molecular biology grade water, and stored at <NUM>. Quantitative PCR (qPCR) with the iTaq Universal Probes Supermix (BioRad, Marnes-la-Coquette, France) was used to quantify human Alu sequences with the TaqMan AluYB8 Probe (Thermo Fischer Scientific). Genomic DNA from the different tissues of the implanted rats was amplified side-by-side with DNA from control rats, spiked-in with variable amount of DNA from U87-MG human cells (<NUM>, <NUM>, <NUM> ng; <NUM>, <NUM>, <NUM>, <NUM>, <NUM> pg or no DNA), to build a standard curve. Samples were run in triplicate for <NUM> cycles on a CFX System (Bio-Rad). The limit of detection corresponded to the average signal from control rat DNA not spiked-in with human DNA.

Four weeks (<NUM> ± <NUM> days) prior to interarticular surgery, adult sheep females (Rideau Arcott Hybrids strain) were subject to bone marrow aspiration procedure. Briefly, animals were placed in ventral recumbency and anesthetized with a mix of glycopyrolate, xylazine and ketamine administered intramuscularly (IM). An IV catheter was placed in the appropriate vein. The larynx was sprayed with lidocaine and the animals were intubated with an appropriate sized cuffed orotracheal tube. If intubation was not possible under IM anaesthesia, induction was performed using isoflurane in O<NUM> (<NUM>-<NUM>%) or propofol intravenously. The sheep were then mechanically ventilated with isoflurane in O<NUM>. The harvest site was disinfected and a needle was introduced in the iliac crest. A sterile <NUM> syringe was filled with <NUM> of <NUM>,<NUM> IU/mL heparin and filled with approximately <NUM> of bone marrow. The syringe containing bone marrow sample and heparin was sealed with an appropriate sterile cap for mesenchymal stem cells isolation, characterization and preparation for the surgical procedure.

A total of <NUM> adult sheep underwent surgical induction of osteochondral defect into the medial femoral condyle. Three groups of sheep (ARTiCAR, AG control, NT control) were considered; each sheep was implanted on either the proximal or distal part of the right or left condyle of posterior legs. For surgery, the hind limb was flexed to a position at which the medial condyle could be palpated under the skin. A <NUM> medial parapatellar skin incision was performed. After blunt dissection of the subcutaneous tissues, the fascia overlying the vastus medialis muscle was incised just distal to the belly muscle with a small incision parallel to the muscle fibers and the vastus was retracted proximally. Blunt dissection was used to expose the periosteum down to the medial condyle of the femur. The joint capsule and periosteum were incised just proximal to the origin of the medial collateral ligament. Overlying soft tissues were removed from the bone only in the vicinity of the drill holes. Holes were predrilled using a <NUM>-mm drill bit to a depth of <NUM>, except for the AG group, where the hole had a depth of <NUM>.

Following the induction of the defect, the NanoM1-BMP2 was placed, and the defect was filled with MSCs/hydrogel mix (ARTiCAR combined ATMPs, n = <NUM>). In the AG group (n = <NUM>), a bone sample of <NUM> of diameter and <NUM> deep was taken out from the condyle and placed into the defect. In the NT group (n = <NUM>) the defect was neither treated, nor filled. Up to <NUM> <NUM>% bupivacaine were infiltrated into the surgical site to achieve local anaesthesia and manage pain after surgery. The tissues were repositioned and closed layer-by-layer using appropriate sutures. Postoperative analgesia and antibiotic therapy were performed, <NUM>/kg Excede (IM) was administered during recovery from anaesthesia, and <NUM>/kg caprofen (IM) was administered <NUM> days after surgery.

For the longitudinal analysis of the knee repair, sheep were examined three times via MRI, immediately after surgery, at <NUM> and <NUM> weeks, using a Magnetom Verio 3T (Siemens). For the procedure, sheep were anaesthetised with an intravenous injection of <NUM>/kg xylazine and <NUM>/Kg ketamine and placed in dorsal decubitus. A total of six sites of surgery were imaged for each group. Proton density weighted, fat-saturated sagittal sections of the acquisitions were analysed using the Osirix opensource software.

For analysis of the bone mineralization, sheep were anesthetized, weighed and euthanised by a lethal injection of <NUM>/ml Euthanyl rapid IV bolus <NUM> weeks after surgery. Death was confirmed and recorded by observation of asystole or ventricular fibrillation, either on the electrocardiogram or by auscultation. Femoral condyle from were explanted from euthanized sheep and imaged via 3D micro-CT (Quantum Fx mCT, Julien Becker, ICS, IGBMC, Strasbourg, France). A total of six sites of surgery were imaged for each group. Three-dimensional surface rendering was obtained from micro-CT 2D images using the Osirix open-source software.

Treated femurs were removed from euthanized animals and subject to macroscopic inspection of the articular surface. The distal femoral epiphysis (with condyles) were individually identified and collected in <NUM>% neutral buffered formalin, after macroscopic examination. Bone blocks were cut in two halves, by sawing in the middle of the sample along its longitudinal axis. Sections were cut through the defect along its deeper axis, from the bone surface to the end of the drill hole producing rectangular-shaped defect half sections. Full-thickness femoral bone-cartilage defect sites underwent undecalcified bone preparation and were infiltrated with methylmethacrylate and polymerized. A single <NUM> section spanning the entire width of the defect was cut along the parasagittal plane from each medial femoral condyle. The sections were stained with safranin o - fast green, for the staining of both cartilage and bone. The femoral defect sites were carefully evaluated and scored according to the ICRS histological score system according to the below Table:
<IMG>.

After either <NUM> or <NUM> weeks from implant, safety parameters were evaluated for ARTiCAR (n = <NUM>-<NUM> at <NUM> weeks; n = <NUM>-<NUM> at <NUM> weeks) and compared to autograft (n = <NUM> at <NUM> weeks; n = <NUM> at <NUM> weeks) treatment (comparable to mosaicplasty currently performed for cartilage treatment in surgery) and to no treatment (n = <NUM> at <NUM> weeks; n = <NUM> at <NUM> weeks). Values are represented as mean ± SD. Differences were evaluated with One-Way ANOVA/Kruskal-Wallis test. * = p ≤ <NUM> between ARTiCAR and autograft.

Also, the tissue underneath and adjacent to the defect was evaluated for a number of parameters (see below table <NUM>) to assess safety and efficacy of the treatments.

After either <NUM> or <NUM> weeks from implant, safety parameters were evaluated for ARTiCAR (n = <NUM>-<NUM> at <NUM> weeks; n = <NUM>-<NUM> at <NUM> weeks) and compared to autograft (n = <NUM> at <NUM> weeks; n = <NUM> at week <NUM>) treatment (comparable to mosaicplasty currently performed for cartilage treatment in surgery) and to no-treatment (n = <NUM> at week <NUM>; n = <NUM> at week <NUM>). Values are represented as mean ± SD. NA: not applicable, phf*: per high powered (x400) field.

Results from the WST1 assay were statistically evaluated using <NUM>-Way ANOVA followed by Tukey post-hoc test on Prism <NUM> (GraphPad). A p value ≤ <NUM> was considered significant. One-way ANOVA followed by Bonferroni post-hoc test was used to compare haematological and biochemical parameters in the blood tests, using Prism <NUM>. A p value ≤ <NUM> was considered significant. Both SigmaPlot (SYSTAT Software) and Prism <NUM> (GraphPad) were used to compare the ICSR II scores from the in vivo experiments in sheep. Equal variance test and normality tests were performed. Either one- (differences induced by treatment) or two-way ANOVA (differences induced by both treatment and time) followed by Bonferroni post-hoc test were used to assess significant differences among the continuous variables of the study groups. If either equal variance test or normality test failed, a Kruskal-Wallis one-way ANOVA with Dunn's correction was conducted. A p value ≤ <NUM> was considered significant.

The nanofibrous PCL wound dressing component for the bone wound patch (c) (NanoM1-BMP2; <FIG>) of the ARTiCAR combined ATMPs was tested for cytotoxicity on MRC-<NUM> foetal lung fibroblasts in vitro. Cells were seeded in the presence of the NanoM1-BMP2 wound dressing and compared to positive (polyurethane film; RM-A) and negative (high density polyethylene film; RM-C) controls. Different sizes of both the NanoM1-BMP2 membrane and the control films were tested in the range of <NUM>-<NUM><NUM>. Cell density and morphology were qualitatively evaluated by bright field microscopy. Cells cultured in the presence of RM-A started to detach already after <NUM> hours (<FIG>). One the contrary, cells cultured in the presence of the NanoM1-BMP2 scaffold did not show any morphological abnormalities (<FIG>), as they did those cultured in the presence of RM-C films (<FIG>). Next, we assessed the viability of the MRC-<NUM> cells in the <NUM> conditions tested, using the WST-<NUM> live/dead cell assay. Both RM-A and NanoM1-BMP2 showed a decrease in cell viability over <NUM> hours that was directly proportional to the size of the membrane used, as the interpolated trend lines indicated (solid black lines in <FIG>). However, in the presence of <NUM><NUM> RM-A, the cell viability reduced to <NUM>± <NUM>% compared to t0 (<FIG>, p ≤ <NUM>), while in the presence of a fragment of NanoM1-BMP2 of the same size, the cell viability reduced to <NUM> ± <NUM>% (<FIG>). No significant reduction of the cell number was also observed in the presence of the negative control film (<FIG>). These results indicate that the NanoM1-BMP2 is not toxic to MRC-<NUM> cells in vitro.

The acute toxicity of the ARTiCAR was evaluated in vivo in nude rats, and compared to the non active part of the implant (hydrogel without hMSCs) as a vehicle. Clinical, haematological and biochemical parameters were evaluated. The biodistribution and the persistence of the transplanted cells were also assessed. Briefly, ARTiCAR combined ATMPs (group <NUM>) or vehicles (group <NUM>) were implanted into femoral defects in nude rats. Ventricular blood was taken before the animals were euthanised, <NUM> days post implant, and femurs were collected for histopathology analysis. Neither the ARTiCAR combined ATMPs, nor the vehicle triggered any significant effect on the body weight, either in female or male rats, over a period on <NUM> days following the implant (<FIG>). Haematological parameters (<FIG>). showed no significant differences among the <NUM> groups of animals (group <NUM> male, group <NUM> female, group <NUM> male, group <NUM> female). Biochemical parameters were also assessed (<FIG>). Female rats in Group <NUM> showed significantly higher plasmatic concentrations of both Alanine aminotransferase (ALAT; <NUM> ± <NUM> U/I vs. <NUM> ± <NUM> U/I for group <NUM> and <NUM>, respectively; p ≤ <NUM>) and Calcium (<NUM>± <NUM>/l vs. <NUM> ± <NUM>/l for group <NUM> and <NUM>, respectively; p ≤ <NUM>) than those in group <NUM>. These differences were not associated with any additional symptoms and, altogether, the analysis of the haematological and biochemical parameters considered did not show any clinically relevant differences between ARTiCAR-treated animals and the control group. The femur-tibia joints were also collected and subject to histological analysis. Both the ARTiCAR and the vehicle induced comparable levels of inflammatory response at the implant site (delimited by asterisks in 3D,E), compatible with the bone healing process of the induced bone defect (<FIG>). Eventually, the biodistribution of the human MSCs at day <NUM> post implant was also assessed, using qPCR for detecting human DNA. Signal from hMSCs DNA was never detected above the threshold level, except in the testis of one male rat in group <NUM>. The migration of the PCR product on <NUM>% agarose gel confirmed the specificity of the amplification product. Taken together, clinical, haematological and biochemical data suggest that the ARTiCAR implant did not induce any clinically relevant symptoms; the inflammatory response detected from the histological analysis of the implant site revealed no differences with the control group, strongly indicating the safety of the ARTiCAR implant for the treatment of bone defects.

To further confirm the safety of the ARTiCAR combined ATMPs, and to assess the feasibility of its usage in large animals, osteoarticular defects were induced in femoral condyles of adult sheep and were either left unfilled (no-treatment control: NT) of filled with the ARTiCAR implant or with an autograft (AG). The healing process was monitored non-invasively by means of magnetic resonance imaging (MRI) at <NUM>, <NUM> and <NUM> weeks (<FIG>). After either <NUM> or <NUM> weeks from implant, sheep were euthanized and the femur-tibia joints were explanted and scanned via micro-computing tomography (micro-CT; <FIG>); a 3D surface rendering of the joint was also built from 2D section images (<FIG>). The explanted joints were macroscopically scored according to the ICRS score system as follows: grade I = normal cartilage, grade II = nearly normal, grade III = abnormal cartilage and grade IV = severely abnormal cartilage (<FIG>). Eventually, the explants were stained in a solution of safranin o - fast green and examined histologically (<FIG>). The following parameters were taken into consideration within the repaired tissue and scored according to ICRS II score system: subchondral bone abnormalities/marrow fibrosis, tissue morphology, cell morphology, basal integration, formation of a tidemark, vascularization, overall assessment and mid/deep zone assessment (table <NUM>). In general, a correct subchondral bone formation, a proper osteochondral remodeling zone and a good integration between graft and host tissues were observed within the induced bone defect <NUM> weeks after treatment in all the experimental groups considered (<FIG>). Interestingly, <NUM> weeks post implant, the ARTiCAR-treated defects showed reduced vascularization (<NUM> ± <NUM>%), poorer quality of the subchondral bone (<NUM> ± <NUM>%) and reduced tidemark (<NUM> ± <NUM>%) (<FIG>, left panels). These scores were aligned to the NT group (<NUM> ± <NUM>%; <NUM> ± <NUM>%; <NUM> ± <NUM>%, respectively), but significantly different to the AG group (<NUM> ± <NUM>%, p ≤ <NUM>; <NUM> ± <NUM>%, p ≤ <NUM>; <NUM> ± <NUM>%; p ≤ <NUM>, respectively). However, the ARTiCAR group showed a better vascularization (<NUM> ± <NUM>) and a higher degree of fibrosis (<NUM> ± <NUM>) then the AG groups in the tissues adjacent to the defect (p ≤ <NUM> for both parameters), but not a level of fibrocartilage formation as high as in the NT control (p ≤ <NUM> and p ≤ <NUM> at <NUM> and <NUM> weeks post implant, respectively) (<FIG>, right panels, black arrow in <FIG>). These results suggest that the ARTiCAR might induce a low inflammatory response that in turn triggers regeneration38, inducing subchondral bone formation (yellow arrow in <FIG>) and promoting effective healing in the long term. Moreover, at <NUM> weeks post implant, matrix staining, surface/superficial assessment, mid/deep zone assessment and overall assessment showed the highest scores for the ARTiCAR group (table <NUM>), and no polymorphonuclear cells, infection, fibrinous exudates and fatty infiltrates were detected in the tissue adjacent to the defect in both the ARTiCAR and the AG groups (table <NUM>), indicating that the ARTiCAR combined ATMPs treatment has a safety over the long term comparable to that of an autograft.

In conclusion, the global cartilage repair/regeneration market is valued at USD <NUM> billion in <NUM> and is expected to grow at a CAGR of <NUM>% during the <NUM>-<NUM> period, owing to the increased life expectancy. Currently, the international standard treatment for OA is total knee arthroplasty (TKA). Despite its rapidly increasing utilization (<NUM>,<NUM> patients/year younger than <NUM> years, in the US), TKA is suboptimal in young, physically active patients, as it induces fibrocartilage formation, cellular hyper- or hypotrophy and the lack of a proper interface between cartilage and subchondral bone. Regenerative nanomedicine combines the use of biomaterials, nanotechnologies and cells to offer better solutions to issues like OAR, where a complex interface regeneration is required. In this work, we assessed the feasibility, non-invasive monitoring and safety of the ARTiCAR combined ATMPs. Similarly to other smart implantable scaffolds that promote osteochondral differentiation, the ARTiCAR releases BMP2. However, thanks to the nanoreservoir technology that provide cell contact-dependent, spatial-temporal release, the total amount of BMP2 used in ARTiCAR is <NUM>,<NUM> times lower than that of BMP2-soaked collagen membranes used in the clinic (<NPL>)), reducing both potential inflammatory side effects (<FIG>; <FIG>) and the overall costs of the procedure. Differently to other approaches where a poor subchondral bone regeneration was achieved (<NPL>); and <NPL>)), the ARTiCAR address simultaneous regeneration of both the subchondral bone and the cartilage (<FIG>), representing an innovative technology for promoting OAR in a localized osteochondral defect. For cartilage regeneration, the ARTiCAR incorporates MSCs. Human MSCs are currently used in clinical trials for promoting OAR, because of their transdifferentiation potential coupled to immunomodulatory effect. However, since tumorigenicity of MSCs is debated, the biodistribution of hMSCs is a critical concern of preclinical safety (<NPL>); and <NPL>)). After the implant of ARTiCAR, traces of hMSC DNA were found in the testes of one male nude rat, out of <NUM> implanted animals (<FIG>), highlighting the safety of the ARTiCAR implant.

Claim 1:
A biomaterial comprising:
- a membrane wound patch (a), made of a nanofibrous polymeric scaffold, wherein optionally said polymeric scaffold has a surface being coated
. either with an interrupted coating made of multilayered droplets, said multilayered droplets being droplets composed of at least one layer pair consisting of a layer of polyanions including at least chondroitin sulfate and/or hyaluronic acid, and a layer of polycations, and,
. or with a continuous coating comprising more than <NUM> layer pairs, each layer pair consisting of a layer of polyanions including at least chondroitin sulfate and/or hyaluronic acid, and a layer of polycations;
- a hydrogel (b) including stem cells, and
- a bone wound patch (c) being a nanofibrous scaffold made of polymers, wherein said scaffold has a surface coated with an interrupted coating made of multilayered droplets, said multilayered droplets being droplets composed of at least one layer pair consisting of a layer of polyanions and a layer of polycations, and wherein the bone wound patch (c) further comprises a bone growth factor;
wherein the hydrogel (b) is included between the membrane wound patch (a) and the bone wound patch (c).