The present invention relates to a biomimetic material used for the preparation of mono-, bi- or multi-layer structures for bone, cartilaginous or osteocartilaginous tissue regeneration. The invention also relates to a method for preparing such biomimetic material.

As far back as the mid 18th century it was known that "the articular cartilage, once damaged, has a poor capacity to heal" (Hunter, 1743) . From a biological point of view, articular cartilage is a highly differentiated tissue, whose primary function consists in absorbing the load forces acting upon it and transmitting and distributing them to subchondral bone (L.A. McMahon et al . Regen Med 2008 ; 3 ( 5 ) : 743-759 ) . The smooth, lubricated surface allows low friction between articular heads, thus facilitating the movements thereof. It has a thickness varying between 1 and 7 mm, and consists of several layers with a different composition, functions and properties. Articular cartilage is made up of cells (chondrocytes), which account for 2% of the total volume, immersed in an extracellular matrix consisting of two principal phases: water, which represents 60% of the total weight, and the solid phase (composed of type II collagen in a percentage of 15-22%, other collagens, e.g. type VI, IX, XI, in a percentage <2% and proteoglycans in an amount varying from 4-7%) , which forms an amorphous, isotropic matrix organized in an optimal manner to bear compressive stresses (D.W. Jackson et al . J Am Acad Orthop Surg 2001;9:37-52). Articular cartilage is a non-vascularised, hypocellular tissue and, therefore, following a trauma or degenerative pathology, the resulting tissue defect is healed through the formation of a fibrocartilaginous tissue, characterised by inferior biomechanical and biological properties compared to the native hyaline cartilage (J. Carey-Beth et al . Journal of Athletic Training 2001 ; 36 ( 4 ) : 413-419 ; E.B. Hunziker. Osteoarthritis and Cartilage 2001;10:432-463). Based on the degeneration of the superficial layer, four stages of cartilage damage of increasing severity are recognised: grade 1, in which the cartilaginous mantle appears continuous but with a soft consistency and sometimes swelling; grades 2 and 3, in which the fissures extend completely or almost completely through the thickness of the cartilaginous mantle; grade 4, in which the cartilage is completely eroded and leaves the underlying subchondral bone exposed (I.M. Khan et al . Europ Cells Materials 2008;16:26-39).

With particular reference to osteochondral pathology affecting the knee joint, surgical replacement, i.e. with a metal prosthesis, is the approach generally used in cases of very severe tissue degeneration and as a rule in elderly patients. In young patients and in cases of limited degeneration, a reconstructive surgical approach is preferred instead: classic concepts of mechanical axis realignment can be combined with the new concepts of regenerative medicine and reconstruction of the damaged articular elements. This therapeutic approach is possible thanks to the use of osteochondral substitutes, be they of biological origin, as in the case of autologous, homologous or heterologous transplants, or of synthetic origin, with the function of matrixes which structurally support and favour guided tissue regeneration (I. Martin et al . Journal of Biomechanics 2007;40:750-765).

According to the type and severity of the cartilaginous lesion (superficial, deep or with involvement of the subchondral bone), the following surgical approaches are presently used:

- cartilage shaving (or debridement), which consists in cleaning the focal articular cartilage lesion by shaving of the malacic tissue. This can be done either by means of the "open" arthrotomy approach or arthroscopically . The tissue that subsequently regenerates itself does not possess, however, the hydrophilicity and elasticity characteristic of native cartilage and hence does not satisfy the biological and mechanical needs of the joint. This technique is indicated for lesions of small dimensions and not full-thickness ones (grades 1, 2 and grade 3 only in selected cases);

curettage, which consists in cleaning the focal lesion by completely removing the damaged or diseased tissue, either arthrotomically or arthroscopically . This technique has the aim of eliminating the possibility of articular blockage, decreasing swelling and relieving pain, at least for a certain period of time. In this case as well, the cartilage repair tissue is of the fibrocartilaginous type and thus has inferior properties compared to the native tissue. This technique is indicated for full-thickness lesions (grades 3-4), but only of small dimensions ;

- microfracture surgery, a technique which associates curettage and/or debridement with the drilling of holes in the subchondral bone, achieved with the aid of special awls. This induces bleeding in the bone bed underlying the removed damaged tissue, so that the mesenchymal progenitor cells originating from bone marrow can colonise the osteocartilaginous defect, proliferate, and differentiate into chondrocyte and osteocyte, thus restoring both the chondral and bone tissue. The repair tissue that is generated is of the fibrocartilaginous type and lacks the resistance and effectiveness of the original hyaline cartilage existing prior to the insult. This technique is indicated for full-thickness lesions (grades 3-4), with dimensions of up to 1.5 - 2 cm ² ;

mosaicplasty, a technique consisting in the use of the patient's own cartilage to repair the damaged region. This procedure implies the use of cylinders of autologous osteochondral tissue drawn from non-weight-bearing regions of the articular surface and positioned so as to replace the malacic tissue until the entire damaged area is covered. Mosaicoplasty is indicated for full-thickness cartilage lesions (grades 3 and 4), but of only small and medium size (ideally defects with dimensions of 1 - 4 cm ² ) . Though it is able to reconstruct hyaline-like cartilage in the repaired site, this technique may provoke the onset of morbidity at the donor site, leading to the occurrence of greatly invalidating arthrosic pathologies ;

osteochondral allograft , which consists in the use of cadaveric donor osteochondral grafts supplied by Musculoskeletal Tissue Banks. They have been used in clinical practice for many years for complex articular reconstruction and for the treatment of severe osteochondral lesions (grade 3 and 4) . The biggest problems associated with this approach are represented by the non-constant and non-guaranteed availability of material from the Banks and the possibility of residual risks of pathogen transmission from donor to recipient;

autologous chondrocyte implantation (ACI) , which involves a first intervention, during which a healthy fragment of cartilage is sampled, by arthroscopy, from a non-weight-bearing articular region of the knee, and subsequently treated in the laboratory. The chondrocytes derived from the cartilaginous fragment are expanded in the laboratory for about 30-40 days and the procedure subsequently continues with the second intervention: arthroscopic curettage and/or debridement of the damaged cartilage is performed to render the articular surface suitable for receiving the implantation of expanded cells, injected beneath a periosteal flap. The technique thus involves two surgical interventions and the removal of a fragment of periosteum from the donor site (generally, the proximal tibial metaphysis), with consequent morbidity of the site itself. Moreover, this treatment is applicable only for superficial chondral lesions (grades 1 and 2), but not for lesions involving underlying bone tissue;

matrix-induced autologous chondrocyte implantation (MACI) , which represents an evolution of the ACI technique, as it involves seeding chondrocytes expanded in the laboratory onto specific biomaterials with the aim of favouring the healing of cartilaginous lesions without the aid of a periosteal cover flap. Both techniques, ACI and MACI, have demonstrated to be able to reconstruct a hyaline-like cartilaginous tissue, insofar as regards the treatment of grade 1 and grade 2 cartilage lesions, but they remain non cost-effective procedures, in that they are characterised by two surgical interventions, a stage of cellular expansion in the laboratory for around 30-40 days and sampling from a donor site with a high risk of associated morbidity.

For the treatment of broad osteochondral defects, it has been proposed to use scaffolds or three-dimensional structures capable of mimicking the entire osteochondral compartment, characterised by a well-defined composition, porosity, architecture and biomechanical properties (J.K. Sherwood et al . Biomaterials 2002 ; 23 ( 24 ) : 4739-4751 ; M.M. Stevens. Materials Today 2008;11:18-25). The outer superficial portion (chondral) generally consists of a polymeric layer, whereas the inner portion (subchondral osseous) consists of a composite polymeric material which incorporates an inorganic mineral component.

Thanks to the enormous progress achieved in the field of regenerative medicine and tissue bioengineering, a series of biomimetic materials have recently been developed which are defined as "intelligent", i.e. able to replicate both the chemical composition (chemical biomimetics ) , and architecture (physical biomimetics) of natural cartilage and bone. These matrixes are commonly defined as "biologically inspired materials", that is biomaterials which are produced drawing inspiration from natural biological tissues, synthesized in conditions such as to reproduce a structure and environment wholly similar to physiological ones, and thus able to favour regeneration rather than tissue repair (A. Tampieri et al . Key Engineering Materials 2008:361-363:927-930; A. Tampieri et al . Cartilaginiform and osteochondral substitute comprising a multilayer structure and use thereof. Patent PCT W02006 / 092718 ) . In light of this new concept of tissue regeneration mediated by "intelligent" biomimetic scaffolds, the problem at the basis of the present invention is to provide chondral, osseous or osteochondral substitutes which are alternatives to, or, in any case, endowed with improved characteristics compared to the known ones. In particular, the problem of the invention is to provide chondral, osseous or osteochondral substitutes endowed with suitable biomechanical stability, biocompatibility and degradability, which can be effectively integrated with native tissues surrounding the lesion and promote complete tissue regeneration without evoking or inducing any inflammatory and/or immune response on the part of the human recipient body.

Such problem is solved by a biomimetic material used as a structure for bone, cartilaginous or osteocartilaginous tissue regeneration as described in the appended claims. The problem is also solved by a method for producing the biomimetic material as specified in the appended claims.

Further characteristics and advantages of the present invention will become more apparent from the following detailed description of the invention, made also with reference to the appended drawings in which:

Figure 1 shows a schematic perspective view of a bilayer biomimetic material according to one embodiment of the invention;

Figure 2 contains a graph which shows the results of comparative swelling experiments: bilayer biomimetic material prepared with the three-phase method of the invention compared with bilayer biomimetic material prepared with the known method of physical blending;

Figure 3 shows a SEM photograph of a biomimetic material consisting solely of type I collagen;

Figure 4 shows a SEM photograph of a biomimetic material consisting solely of chitosan;

Figure 5 shows a SEM photograph of the cartilaginous layer of a bilayer biomimetic material prepared with the three-phase method of the invention;

Figure 6 shows a SEM photograph of the cartilaginous layer of a bilayer biomimetic material prepared with the known method of physical blending;

Figure 7 shows a SEM photograph of the osseous layer of a biomimetic material prepared with the three-phase method of the invention; Figure 8 shows a SEM photograph of the osseous layer of a biomimetic material prepared with the known method of physical blending;

Figure 9 shows a comparison of the results of TGA analyses of a cartilaginous layer of bilayer biomimetic materials prepared, respectively, with the three-phase method of the invention and by physical blending;

Figure 10 shows a comparison of the results of the TGA analyses of an osseous layer of bilayer biomimetic materials prepared, respectively, with the three-phase method of the invention and by physical blending;

Figure 11 shows a comparison of the results of the TGA analyses of the bilayer biomimetic materials in toto prepared, respectively, with the three-phase method of the invention and by physical blending;

Figure 12 contains a graph which shows the results of comparative experiments relative to testing of the water solution uptake rate: bilayer biomimetic material prepared with the three-phase method of the invention compared with physical blending.

The present invention relates to a mono-, bi- or multilayer biomimetic material used as a chondral, osseous or osteochondral substitute, obtained by means of a process of synthesis called "pH-dependent three-phase", described in detail further below in the patent application.

The biomimetic material of the invention comprises at least one layer comprising collagen and chitosan. The material is characterised by a degree of equilibrium swelling of between 1450 and 1700%, following immersion in physiological solution at 37 °C, and by a single phase transition in the temperature interval between 200 °C and 400 °C as determined by thermogravimetric analysis (TGA) .

The degree of swelling is measured by means of the swelling test as described in detail in the experimental part of the present patent application in the paragraph entitled "swelling test".

Such test was conducted according to K. Tuzlakoglu et al . Macromol. Biosci. 2004;4:811-819.

The swelling test consists in immersing samples of biomimetic material in a physiological solution at approximately 37 °C for time intervals of between 2 minutes and 24 minutes. The samples of biomimetic material are removed from immersion in physiological solution and weighed at constant intervals of 2-4 minutes, starting from a minimum of 2 minutes and arriving at a maximum of 24 minutes.

The swelling percentage is calculated with the following mathematic formula:

%S = (Mw-Md) /Md x 100 where : Mw is the weight of the wet sample

Md is the weight of the dry sample.

In this way one determines a percentage swelling curve of the sample as a function of time, from which the degree of equilibrium swelling (percentage value corresponding to the plateau of the curve) can be extrapolated.

The curve is also used to determine the interval of time it takes for the sample to reach said equilibrium.

The thermogravimetric analysis conducted in order to determine the phase transition of the biomimetic material according to the invention is based on the Max Resolution procedure, J. Paya et al. Mettler-Toledo UserCom 2000;1:15-17; Mettler-Toledo Datasheet .

Such analysis is described in detail in the experimental part of the present patent application, in the paragraph entitled "Thermogravimetric analysis (TGA)".

The TGA analysis was performed in an air atmosphere in the temperature interval between 40 °C and 1000 °C, with heating at a speed of 20 °C/min, slowed to 1 °C/min at the points corresponding to the various weight losses of the samples analysed . The biomimetic material of the invention comprising chitosan and collagen is used as a replacement structure for articular cartilage, in particular for the treatment of articular cartilaginous defects or to favour the neoformation of cartilaginous tissue.

In the monolayer biomimetic material the collagen is present in an amount of between 30 and 90%, preferably between 50% and 80% by weight, and the chitosan is present in an amount of between 10 and 70% by weight, preferably between 20 and 50% by weight, relative to the total weight of the polymeric components.

Such material has a thickness of between 1 and 6 mm, preferably between 2 and 4 mm.

In a preferred embodiment the above-described monolayer biomimetic material also comprises hydroxyapatite, in addition to collagen and chitosan.

In the latter case, the material is used as a replacement structure for bone tissue, in particular for the treatment of bone defects or to favour the neoformation of articular subchondral bone tissue.

Preferably, the collagen and the chitosan used in the monolayer biomaterial are in the form of fibres. The hydroxyapatite is advantageously used in the form of nano- and microdimensional crystals or granules intimately placed within the fibrous organic matrix created by the chitosan and by the collagen.

Preferably, the hydroxyapatite granules have dimensions of between 10 lm and 30 nm.

In this particular embodiment, the monolayer biomimetic material comprises hydroxyapatite in an amount of between 50 and 400% by weight, preferably between 70 and 300% by weight, relative to the total weight of the polymeric components.

Such monolayer material has a thickness of between 2 and 8 mm, preferably between 3 and 6 mm.

In a preferred embodiment of the invention the biomimetic material comprises a bilayer or multilayer structure capable of mimicking both the cartilaginous component and the subchondral bone component .

In particular, the biomimetic material comprises at least two layers, preferably two layers, wherein the layer that will be placed in contact with the chondral lesion comprises chitosan and collagen, while the layer that will be placed in contact with the bone lesion comprises chitosan, collagen and hydroxyapatite .

The bilayer osteocartilaginous biomimetic material is monolithic and is formed by the physical union of the two previous monolayer substitutes, one defined as cartilaginous and the other as osseous (Figure 1) .

The bilayer or multilayer osteocartilaginous biomimetic material has a thickness of between 3 and 14 mm, preferably between 5 and 10 mm.

This bi- or multilayer biomimetic material is utilized to prepare cartilaginous and osseous substitutes for the simultaneous treatment of articular osteochondral defects or to favour the neoformation of cartilaginous and subchondral bone tissue .

The mono-, bi- or multilayer biomimetic material utilized as an osseous, cartilaginous or osteochondral substitute acts as a temporary scaffold for the in situ adhesion, proliferation and chondrogenic and/or osteogenic differention of undifferentiated mesenchymal stem and/or progenitor cells originating from the marrow blood contained in the subchondral spongy bone tissue, according to the concept of so-called "guided tissue regeneration" (E.T. Baran et al . J Mater Sci Mater Med 2004 ; 15 ( 2 ) : 161-165 ; A.J. Salgado et al . Macromolecular Bioscience 2004; 4:743-765; I.C. Bonzani et al . Current Opinion in Chemical Biology 2006;10:568-575).

Therefore, to favour the regeneration of bone and/or chondral tissues, the biomimetic material of the invention may comprise peripheral blood, marrow blood in toto, marrow blood concentrate, platelet concentrate (PRP) , growth factors or in general factors capable of favouring and promoting cellular adhesion, proliferation and differentiation, such as b-FGF, TGF, IGF-1 and -2, PDGF , EGF , VEGF , BMP etc. Other biological materials that can be combined with the biomimetic material of the invention to favour bone and/or chondral regeneration are undifferentiated mesenchymal stem and/or progenitor cells, autologous or maintained in culture in vitro for a period of time necessary for their multiplication and/or differentiation into osteoblasts and/or articular chondrocytes.

The biomimetic material is thus impregnated or mixed with the aforesaid biological materials before being implanted into the connective and/or articular tissues of the patient.

In a preferred embodiment, the mono-, bi- or multilayer biomimetic material can be associated with components present in the cartilaginous structure, for example by impregnation or exterior coating or physical blending with hydrophilic biopolymers, such as hyaluronic acid and derivatives (cross- linked, esters, sulphated) , keratan sulphate, condroitin sulphate, sodium alginate, gellan, gelatin, elastin, resilin and derivatives .

Such addition makes it possible to improve the properties of biocompatibility, flexibility and hydrophilicity of the biomimetic material and hence stability in the anatomical site of application.

Alternatively, or in addition, the mono-, bi- or multilayer material can be associated with bone tissue-like components: polylactic acid (PLA) , L-polylactic acid (PLLA) , polyglycolic acid (PGA) , polylactic-co-glycolic acid (PLLGA) , polycaprolactone (PCL) , polyethylene glycol (PEG) , gellan, alginates, pectin, elastin, resilin and derivatives, in order to improve its flexibility and mechanical properties; demineralised bone matrix of human (hDBM) or heterologous (eDBM) origin, in order to improve its osteoinductive potential; or tricalcium- phosphate (oc-TCP, β-TCP) or octacalcium phosphate (OCP) , in order to increase its osteoconductive potential.

In a preferred embodiment of the mono-, bi- or multilayer substitutes, use is made of ultraviolet (UV) radiation and/or cross-linking agents, such as glutaraldehyde, formaldehyde, genipin, ethers, bis-epoxides (e.g.: 1.4 butanediol-diglycidyl ether - BDDGE) and hyaluronic acid and derivatives, to impart greater mechanical and viscoelastic properties.

The cartilaginous, osseous or osteochondral substitutes of the invention can be: moulded and adapted according to the dimensions of the cartilaginous, bone and/or osteochondral damage; applied using either arthroscopic or arthrotomic techniques; fixed or not fixed with the aid of absorbable and non-absorbable sutures, surgical glues of biological or synthetic origin; loaded with pharmacologically active ingredients, such as anti-inflammatory corticosteroids, FANS, immunosuppressants, antibiotics, antiblastics , antiproliferatives and antivirals.

The biomimetic material of the invention is endowed with adequate mechanical stability in the implantation site, is degradable and bioreabsorbable (with reference above all to the organic polymeric component) and osteointegrable (with reference above all to the inorganic calcium-phosphatic component).

In the context of the present invention, the term "collagen" means a fibrous structural protein which represents the major component of the extracellular matrix of the connective tissues of the human body. Type I collagen represents the most abundant type of collagen in nature, a major constituent of numerous adult connective tissues, namely, skin, bone, tendons, ligaments and cornea. The collagen used in the present invention can be type I, type II or mixtures of the same, with possible additions of type V, VI, IX and XI collagen; it can originate from different sources, for example, be of bovine, porcine, equine or synthetic (recombinant) origin.

The use of chitosan to prepare the biomimetic chondral, osseous and osteochondral substitutes of the invention derives from the known haemostatic properties thereof (Rao S.B. et al . Journal of Biomedical Materials Research 1997 ; 34 ( 1 ) : 21-28 ; Harish Prashanth K.V. et al. Trends in Food Science & Technology 2007;18:117-131) and from the fact that the peculiar structure of this polysaccharide is chemically similar to that of the proteoglycans of bone tissue, and the glucosamine glycans and hyaluronic acid present in the extracellular matrix of articular cartilage (Jong Eun Lee et al . Tissue Engineering and Regenerative Medicine 2005 ; 2 ( 1 ) : 41-49 ) .

The term "hydroxyapat ite" means, in general, both stoichiometric hydroxyapat ite Caio (P0 ₄ ) ₆ (OH) ₂ , with a high or low degree of crystallinity , and hydroxyapat ite having chemical replacements, enriched with Magnesium (Mg ²⁺ ) , Carbonate (CC>3 ^2~ ) , Citrate (C ₆ H ₅ 0 ₇ ^~ ), Strontium (Sr ²⁺ ) and/or Silicon (Si ⁴⁺ ) ions.

The term "chitosan" means a partially acetylated polyglucosamine, obtained by incomplete deacetylat ion of chitin. It is a hydrophilic copolymer of the units ( 1—>4 ) -2-amine-2- deoxy--D-glucopyranosyl and ( 1—>4 ) -2-acetamide-2-deoxy--D- glucopyranosyl , with statistical distribution of the two units within the polymeric chain and proportion determining the degree of acetylation. The chemical formula of chitosan is (C ₆ H ₁₁ O ₄ ) _n , with a molecular weight of between 50 and 300 KDa, degree of acetylation < 30%, of animal (insects, arthropods, mollusks, crustaceans) or preferably microorganismal (bacteria, yeast) origin .

The biomimetic material comprising at least one layer is realized by means of a pH-dependent three-phase process of synthesis, comprising the steps of:

a) preparing an acidic collagen suspension at a temperature of between 30 and 45 °C;

b) bringing the pH of the collagen suspension to a value below the isoelectric point of collagen by addition of a basic solution;

c) bringing the pH of the collagen suspension of step b) to a value above the isoelectric point of collagen but below the isoelectric point of chitosan by addition of a chitosan solution or of a chitosan-hydroxyapat ite suspension;

d) bringing the pH of the chitosan-collagen or chitosan- collagen-hydroxyapat ite suspension of step c) to a value above the isoelectric point of chitosan by addition of a basic solution or of a basic buffer;

e) allowing the collagen-chitosan or collagen-chitosan- hydroxyapatite suspension to mature until the final product is obtained.

The pH-dependent , three-phase process of synthesis of the biomimetic material comprising at least one layer may, if necessary, further comprise the step of:

f) submitting the chitosan-collagen or chitosan-collagen- hydroxyapatite suspension of step e) to chemical cross-linking by addition to the suspension of a cross-linking agent.

Furthermore, the process may conclude with the lyophilization of the chitosan-collagen or chitosan-collagen-hydroxyapatite layer and/or packaging and sterilization (step g) .

In a preferred embodiment, the process for preparing the biomimetic material comprises a step h) wherein at least one layer comprising chitosan-collagen according to step e) or step f) and at least one layer comprising chitosan-collagen- hydroxyapatite according to step e) or step f) are physically positioned one on top of the other and allowed to rest until a monolithic bilayer or multilayer structure is obtained through the action of gravity.

Preferably, the bilayer or multilayer structure obtained in step h) is submitted to lyophilization and/or packaging and sterilization (step i)).

The collagen suspension of step a) is prepared by heating the polymer in a solution of organic acid, preferably acetic acid, for a time ≤ 20 minutes and at a temperature lower than that of collagen denaturation .

The pH variation of step b) is obtained by adding to the collagen suspension an aqueous solution of a strong base, preferably sodium hydroxide, up to a pH ≤ 5. The addition of the strong base takes place by slow dripping.

The collagen-chitosan or collagen-chitosan-hydroxyapatite suspension of step c) is obtained by adding to the collagen suspension of step b) a chitosan or chitosan-hydroxyapatite suspension so as to obtain a pH of between 6 and 7, preferably between 6 and 6.5, in the case of the chitosan suspension, or preferably between 6.5 and 7 in the case of the chitosan- hydroxyapatite suspension.

The addition is effected by slow dripping of the chitosan or chitosan-hydroxyapatite suspension .

The chitosan-hydroxyapatite suspension is obtained by adding hydroxyapatite powder to a chitosan solution so that said mixture reaches a pH value above the point of hydroxyapatite dissolution. Preferably the pH of the chitosan-hydroxyapatite suspension is between 6 and 7, preferably between 6.5 and 7.

The pH variation of step d) is obtained by adding to the collagen-chitosan or collagen-chitosan-hydroxyapatite suspension an aqueous solution of a strong base, preferably sodium hydroxide, or a basic buffer solution, preferably a carbonate buffer .

The suspension thus obtained is maintained under stirring for a time of between 5 and 20 minutes, preferably between 5 and 10 minutes .

Maturation of the chitosan-collagen or chitosan-collagen- hydroxyapatite suspension takes place for a time of between 30 minutes and 2 hours, preferably between 30 minutes and 1 hour. The cross-linking of the collagen-chitosan or collagen-chitosan- hydroxyapatite suspension of step f) is preferably effected after washing with water and sieving of the suspension. The cross-linking is preferably conducted using the cross-linking agent BDDGE .

The pH of the cross-linking step is between 8 and 11; the temperature is between 0 and 40 °C; the reaction time is between 24 and 96 hours.

The lyophilization phase of step g) and step i) is preferably preceded by washing with water and sieving. The lyophilization is conducted by bringing the suspension to a temperature between -30 and -40 °C, while submitting it to a vacuum of between 0.1 and 1 mbar, preferably between 0.2 and 0.6 mbar . The suspension is then heated at a temperature of between 20 and 30 °C while being maintained under vacuum.

Sterilization of the finished mono-, bi- or multilayer product is preferably conducted by gamma radiation.

The subject of the invention is a biomimetic material comprising at least one layer comprising chitosan-collagen or chitosan- collagen-hydroxyapatite, obtainable with the above-described method of preparation.

A further subject of the invention is a biomimetic material comprising at least two layers, whereof at least one comprises chitosan-collagen and at least another comprises chitosan- collagen-hydroxyapatite, obtainable by means of the above- described process. In a preferred embodiment, the biomimetic material obtainable by means of the above-described process comprises due layers (bilayer) , whereof one layer comprises chitosan-collagen and the other layer comprises chitosan- collagen-hydroxyapatite .

The above-described method is a three-phase progressive process of fibrating the collagen-chitosan or collagen-chitosan- hydroxyapatite mixtures. The addition, to the collagen suspension, of a chitosan solution or suspension having a pH of between 6 and 7 results in the exceeding of the isoelectric point of the collagen and the precipitation thereof, with the consequent partial incorporation of the chitosan itself into the collagen fibres undergoing maturation. Subsequently increasing the pH to values equal to or greater than 9 brings about the complete maturation of the collagen fibres:

^■ phase I —> pH < isoelectric point of collagen = early precipitation of collagen ( subfibrils ) ,

^■ phase II —> pH > isoelectric point of collagen and < isoelectric point of chitosan = complete precipitation of collagen (fibrils) and incorporation of chitosan (which may or may not be combined with hydroxyapatite ) ,

^■ phase III -^ H > isoelectric point of chitosan = complete maturation of collagen (fibres) and coating thereof with residual chitosan fibres/aggregates (chitosan deposition). The three-phase fibrating process allows the complete integration and physicochemical interconnection of collagen and chitosan, resulting in biomimetic materials consisting of mixed fibres and laminae with an orderly morphology and characterised by superior hydrophilism (water uptake/swelling), adaptability and spatial stability in the implantation site, homogeneity/structural orderliness and shape memory (elastic return under manipulation) compared to scaffolds realized with the known method of simple physical coupling (physical blending) of collagen and chitosan or collagen, chitosan and hydroxyapatite .

As regards the mixing of collagen and chitosan (or of collagen and chitosan-hydroxyapatite ) at a pH close to neutrality, between 6 and 7, the physical combination of collagen and chitosan gels at an acidic pH has been reported in the literature and subsequently the precipitation thereof is favoured by increasing the pH with NaOH solutions (physical blending) . Under the latter conditions, however, it can be assumed that the precipitation of the collagen fibres will tend to be sterically hindered by the presence of chitosan and that one will obtain substitutes having a more irregular, disorderly and inhomogeneous structure, as well as inferior physicochemical performances compared to the three-phase process of the invention .

The use of a chitosan solution in acetic acid/sodium acetate, at a pH of between 6 and 7, preferably between 6.5 and 7, favours the fibrating process (as reported in the experimental part) . The acetic acid/sodium acetate pair allows pH values close to neutrality to be reached without causing the chitosan solution to become turbid or the initial precipitation-aggregation thereof. In fact, an acidic chitosan suspension would generally tend to become turbid and precipitate, thus raising the pH to values between 6 and 7 and precluding a homogeneous mixing with the collagen suspension. Furthermore, its combination with hydroxyapatite creates a uniform suspension whose pH, greater than the dissolution pH of hydroxyapatite itself, does not undergo an increase such as to affect the efficacy of the three- phase progressive fibrating process.

The use of a carbonate buffer solution or NaOH to slowly increase the pH of the collagen-chitosan or collagen-chitosan- hydroxyapatite mixtures to values between 9 and 9.5 favours the fibrating process. At these pH values, in fact, complete maturation of the collagen fibres is obtained.

EXPERIMENTAL PART

Collagen preparation

The collagen [(e.g. type I collagen of equine origin (Baxter Healthcare Corp., Opocrin S.p.A., etc.), bovine origin (Southern Lights Bio, Devro Medical Ltd, Sigma-Aldrich, etc.) or porcine origin (SunMax Biotechnology Co. Ltd, ColBar Life Science Ltd, Nitta Gelatin, Inc., etc.)] is available on the market as a standard product. After the purification process, it is dissolved in a calculated volume of acetic acid (1% in water) until a homogeneous suspension (concentration of between 0.5 and

2%, preferably 1%) having an acidic pH, preferably < 3.5, is obtained. Isoelectric point of collagen, pH=5.5; denaturation of collagen, T ≡ 54 °C.

Chitosan preparation

Chitosan is available on the market as a product of animal origin (e.g. produced by Sigma-Aldrich, Hawaii Chitopure, Norwegian Chitosan, Altakitin, etc.) or microorganismal origin (preferably extracted from yeast, e.g. produced by KitoZyme) , in powder form. After solubilization in a calculated volume of acetic acid (1% in water), the chitosan suspension is combined with sodium acetate (30% in water) until a homogeneous mixture (concentration of between 1 and 4%, preferably 2%) having a pH of between 6 and 7, preferably between 6.5 and 7 (chitosan solution) is obtained. Isoelectric point of chitosan, pH=8.7. Hydroxyapatite preparation

The hydroxyapatite (e.g. produced by Sigma-Aldrich, Merck Co., Berkeley Advanced Biomaterials Inc., Fluidinova, etc.), stoichiometric or enriched (with carbonate, citrate, strontium or silicon ions, preferably magnesium ions), is available on the market as a standard product. Hydroxyapatite dissolution, pH < 5.5.

Preparation of monolayer cartilaginous biomimetic material

The mixing procedures are carried out with the aid of a thermostat magnetic stirrer, rotation speed between 250 and 750 rpm, preferably 500 rpm.

A) 100 g of 1% Collagen in acetic acid is heated at a T lower than that of collagen denaturation, preferably between 25 and 45 °C, for at least 20 minutes, in order to fluidify the polymeric suspension .

B) The pH of the acidic collagen suspension is increased to a value below the isoelectric point of collagen, preferably pH < 5, by slow dripping of 30 cc of an aqueous solution of NaOH 0.1M (pH=13) .

C) 21 g of 2% chitosan solution is added to the collagen suspension, by slow dripping, so as to increase the pH of the collagen suspension to a value above the isoelectric point of collagen but below the isoelectric point of chitosan, pH of between 6 and 7, preferably between 6 and 6.5.

D) The pH of the collagen-chitosan suspension is increased to a value above the isoelectric point of chitosan, pH of between 9 and 13, preferably between 9 and 9.5, by slow dripping of 75 cc of a carbonate buffer solution (1% in water, pH=10) or an aqueous solution of NaOH 0.1M (pH=13) . The suspension is maintained under stirring for a time of between 5 and 20 minutes, preferably between 5 and 10 minutes.

E) The fibrous collagen-chitosan suspension is left to mature in static conditions for a time of between 30 minutes and 2 hours, preferably between 30 minutes and 1 hour.

F) The fibrous collagen-chitosan suspension is washed with water, sieved and submitted to chemical cross-linking by mixing with a 0.5% BDDGE solution in buffer solution (preferably, carbonate buffer) at a pH of between 8 and 11 (preferably, pH=10) and a temperature of between 0 and 40 °C (preferably, 37 °C) for a time of between 24 and 96 hours (preferably, 48 hours ) .

G) The cross-linked fibrous collagen-chitosan suspension is washed with water, sieved and submitted to lyophilization . In particular, the suspension is brought to a temperature of between -30 and -40 °C (preferably, -35 °C) , submitted to a vacuum of between 0.1 and 1 mbar (preferably, between 0.2 and 0.6 mbar) and heated to room temperature (preferably, between 20 and 30 °C) , maintaining the degree of vacuum.

H) The monolayer cartilaginous substitute is packaged in a double pouch and sterilized by γ-radiation at 25 kGy.

Preparation of monolayer biomimetic osseous material

The mixing procedures are carried out with the aid of a thermostat magnetic stirrer, rotation speed between 250 and 750 rpm, preferably 500 rpm.

A) 100 g of 1% Collagen in acetic acid is heated at a T lower than that of collagen denaturation, preferably between 30 and 45 °C, for at least 20 minutes, in order to fluidify the polymeric suspension .

B) The pH of the acidic collagen suspension is increased to a value below the isoelectric point of collagen, preferably pH < 5, by slow dripping of 30 cc of an aqueous solution of NaOH 0.1M (pH=13) .

C) A chitosan-hydroxyapatite suspension is obtained by slowly adding 2 g of hydroxyapatite powder to 16.5 g 2% chitosan solution, via magnetic stirring, so that the pH of the mixture reaches a value above the dissolution point of hydroxyapatite, pH of between 6 and 7, preferably between 6.5 and 7.

D) The chitosan-hydroxyapatite suspension is added to the collagen suspension, by slow dripping, so as to increase the pH of the collagen suspension to a value above the isoelectric point of collagen but below the isoelectric point of chitosan, pH of between 6 and 7, preferably between 6.5 and 7.

E) The pH of the collagen-chitosan-hydroxyapatite suspension is increased to a value above the isoelectric point of chitosan, pH of between 9 and 13, preferably between 9 and 9.5, by slow dripping of 75 cc of a carbonate buffer solution (1% in water, pH=10) or an aqueous solution of NaOH 0.1M (pH=13). The suspension is maintained under stirring for a time of between 5 and 20 minutes, preferably between 5 and 10 minutes.

F) The fibrous collagen-chitosan-hydroxyapatite suspension is left to mature in static conditions for a time of between 30 minutes and 2 hours, preferably between 30 minutes and 1 hour.

G) The fibrous collagen-chitosan-hydroxyapatite suspension is washed with water, sieved and submitted to chemical cross- linking by mixing with a 0.5% BDDGE solution in buffer solution (preferably, carbonate buffer) at a pH of between 8 and 11 (preferably, pH=10) and a temperature of between 0 and 40 °C (preferably, 37 °C) for a time of between 24 and 96 hours (preferably, 48 hours) .

H) The cross-linked fibrous collagen-chitosan-hydroxyapatite suspension is washed with water, sieved and submitted to lyophilization . In particular, the suspension is brought to a temperature of between -30 and -40 °C (preferably, -35 °C) , submitted to a vacuum of between 0.1 and 1 mbar (preferably, between 0.2 and 0.6 mbar) and heated to room temperature (preferably, between 20 and 30 °C) , maintaining the degree of vacuum .

I) The monolayer osseous substitute is packaged in a double pouch and sterilized by γ-radiation at 25 kGy.

Preparation of the bilayer osteocartilaginous biomimetic material

In a preferred embodiment, the collagen-chitosan and collagen- chitosan-hydroxyapatite suspensions are prepared according to steps A) - F) and A) - G) respectively.

J) The cross-linked fibrous suspensions are washed with distilled water, sieved and physically positioned one on top of the other, in such a manner as to create, through the action of gravity, a monolithic bilayer structure characterised by a lower osseous layer and an upper cartilaginous layer. K) The bilayer osteocartilaginous substitute is submitted to lyophilization; in particular it is brought to a temperature of between -30 and -40 °C (preferably, -35 °C) , submitted to a vacuum of between 0.1 and 1 mbar (preferably, between 0.2 and 0.6 mbar) and heated to room temperature (preferably, between 20 and 30 °C) , maintaining the degree of vacuum.

L) The bilayer osteocartilaginous substitute is packaged in a double pouch and sterilized by γ-radiation at 25 kGy.

Physicochemical analyses of the biomimetic material.

The bilayer osteocartilaginous biomimetic material produced by means of the three-phase process described above and shown in Figure 1 was characterised from a physicochemical and mechanical point of view by means of a wettability test (water uptake/swelling, according to K. Tuzlakoglu et al. Macromol. Biosci. 2004;4:811-819), scanning electron microscopy SEM (LEO model 14-30), and thermogravimetric analysis TGA (Mettler-Toledo model TGA/DSC 1, according to B. Benzler. Mettler-Toledo UserCom 2001; 1 : 7, 8; J. Paya et al . Mettler-Toledo UserCom 2000;1:15-17; Mettler-Toledo Datasheet. MaxRes Thermal Analysis from www.mt.com/ta) and a measurement of the water uptake rate.

The same characterisation was performed on an osteocartilaginous material prepared during the same experimental sessions, using the same raw materials in the same proportions by percentage weight, following a protocol analogous to the one described in the previous paragraph, with the sole difference that the single layers (cartilaginous and osseous) were produced by means of a physical mixing procedure at an acidic pH and coprecipitation at a neutral pH (physical blending) as described in the literature (D. Shi et al. Chin Med J 2005 ; 118 ( 17 ) : 1436-1443 ; X. Wang et al . Key Engineering Materials 2007 ; 330-332 : 415-418 ; X. Wang et al . J Biomed Mater Res A. 2009 Jun 15 ; 89 ( 4 ) : 1079-87 ) , rather than by means of the pH-dependent three-phase process of synthesis of the present invention (three-phase method) .

Experimental results

Swelling test The hydrophilism of the osteocartilaginous substitutes prepared with the pH-dependent three-phase process of synthesis (three- phase method) and by coprecipitation (physical blending) was evaluated in triplicate by means of the swelling test (swelling following liquid uptake) , by immersing them in saline physiological solution at 37 °C for various intervals of time and weighing them with an electronic scale, until reaching equilibrium. The degree of swelling was calculated by means of the following formula:

Swelling Percentage = %S = (Mw - Md) /Md x 100,

where :

Mw is the weight of the wet sample,

Md is the weight of the dry sample.

Figure 2 shows the results of the test performed on two representative preparations: the osteocartilaginous substitute obtained by means of the pH-dependent three-phase process of synthesis shows a gradual swelling in the first 8 minutes, reaching equilibrium in 12 minutes; the substitute obtained by physical blending shows a more rapid swelling, reaches equilibrium in 8 minutes, remaining at a distinctly lower mean swelling % (on average, 132% lower) .

The higher swelling % and the slower swelling observed for the osteocartilaginous substitute prepared by means of the three- phase process should guarantee, once it is positioned inside the tissue defect, both a greater mechanical stability in situ, and better adaptation to the specific geometrical characteristics of adjacent tissues.

Scanning electron microscopy SEM

The morphology of the osteocartilaginous substitutes prepared with the pH-dependent three-phase process of synthesis (three- phase method) and by coprecipitation (physical blending) was evaluated by microphotography with a scanning electron microscope, with the aim of revealing any significant differences in terms of homogeneity, porosity, spatial distribution and three-dimensional structure of the collagen and/or chitosan fibres, combined or not combined with hydroxyapatite, according to whether the osseous or cartilaginous layer was concerned.

For this purpose scaffolds consisting solely of collagen (Figure 3) or solely of chitosan (Figure 4) were also evaluated: the former are characterised by laminae with irregular ("jagged") edges and arrangement (Figure 3A, B) , mixed together with fibrous interconnecting structures of varying degrees of complexity and dimensions (subfibrils, fibrils, fibres) (Figure 3C, D) ; the latter, in contrast, show a structure that is mainly characterised by broad parallel laminae with regular ("smooth") edges (Figure 4A,C), linked by slender filaments with triangular insertion (Figure 4D) . The cartilaginous layer of the biomimetic material produced with the three-phase method shows an extremely homogeneous and orderly morphology (Figure 5A) , characterised by broad parallel laminae with irregular edges (Figure 5B) , connected by filamentous fibrous structures of medium dimensions (Figure 5C) : in actual fact, a biomimetic material with an intermediate morphology, arising from the physicochemical interaction and stable integration between collagen and chitosan .

The cartilaginous layer of the biomimetic material produced by physical blending displays a more inhomogeneous and disorderly morphology (Figure 6A) , characterised by the coupling of clearly distinct structures, which can be traced back to those observed in the materials containing only collagen or only chitosan. In particular, both laminae with irregular edges and fibrous structures with varying degrees of spatial organisation (typical of collagen) , and larger-sized laminae with more regular edges can be distinguished (Figure 6B, C) .

The osseous layer of the material produced with the three-phase method shows a regular structure characterised by a homogeneous, uniform distribution of the hydroxyapatite granules (Figure 7A) . The latter are incorporated at varying depths within the organic fibres and laminae (of collagen/chitosan) (Figure 7B) , to such an extent that their edges cannot be clearly distinguished (Figure 7C) ; the more superficial granules, moreover, are clearly coated by a layer of organic material, plausibly chitosan (Figure 7D) .

The osseous layer of the material produced by physical blending reveals a more irregular and disorderly structure characterised by a more inhomogeneous distribution of the hydroxyapatite granules (Figure 8A) , which are not incorporated within the organic component, but are rather mainly resting upon it (Figure 8B, C) . Under greater magnification, it is possible to clearly distinguish the edges of the aforesaid granules (Figure 8D) , which consequently interact in a weaker manner with the collagen and chitosan fibres and laminae. Furthermore, it is clearly observable that, with the same quantity by weight of hydroxyapatite added during the preparation stage, the osseous layer produced by physical blending (Figure 8C) is characterised by a lower density of granules compared to that of the three- phase method (Figure 7B) , further proof of their weak interaction with the organic component of the material.

Thermogravimetric analysis (TGA)

The TGA analyses were performed with two different procedures:

A) in air atmosphere, with a flow rate of 80 ml/min, in a temperature interval between 40 and 1000 °C, heating being carried out at the following velocities (V) (Standard procedure according to B. Benzler. Mettler-Toledo UserCom 2001 ; 1 : 7 , 8 ) :

- 40÷150 °C, V=20 °C/min;

- 150÷700 °C, V=5 °C/min;

- 700÷1000 °C, V=20 °C/min.

B) in air atmosphere, with a flow rate of 80 ml/min, in a temperature interval between 40 and 1000 °C, heating being carried out at a velocity of 20 °C/min, then slowed to 1 °C/min at the point corresponding to the various weight losses of the samples analysed (MaxResolution procedure, J. Paya et al . Mettler-Toledo UserCom 2000;1:15-17; Mettler-Toledo Datasheet. MaxRes Thermal Analysis from www.mt.com/ta).

The standard analysis performed on the cartilaginous layer of the materials produced with the three-phase method and by physical blending (Figure 9A) revealed three weight losses, associated with the loss of water, degradation and subsequent combustion of the organic component (collagen and chitosan) . The analysis in the first derivative (Figure 9A') revealed that the inflection points associated respectively with the loss of water of hydration and degradation of the organic component occurred at higher temperatures in the case of the material prepared with the three-phase method, as compared to physical blending. In the temperature interval between 200 and 400 °C (associated with the degradation of the organic component), the MaxResolution analysis (Figure 9B) showed a single weight loss (ascribable to a sole homogeneous physicochemical structure) in the case of the biomimetic material prepared with the three-phase method, whereas three distinct weight losses (ascribable to a more inhomogeneous physicochemical structure consisting of at least three organic entities with different degradation temperatures) were observed in the case of physical blending.

The standard analysis performed on the osseous layer of the materials produced with the three-phase method and by physical blending (Figure 10A) revealed three weight losses (loss of water, degradation and subsequent combustion of the organic component) and the presence of a final inorganic residue stable at high temperature (corresponding to the hydroxyapatite ) . Said residue was approximately 55% in the case of the material prepared with the three-phase method and approximately 40% in the case of physical blending: as shown by the SEM analysis, this aspect highlights that, with the same quantity by weight of hydroxyapatite added during the preparation stage, the osseous layer produced with the three-phase method, as opposed to physical blending, is characterised by a stronger, more effective interaction between the inorganic and organic components, which means that the material obtained with the three-phase process has an ability to incorporate and hold a larger quantity of the inorganic component (hydroxyapatite ) .

The analysis in the first derivative (Figure 10A') revealed higher temperatures associated with the inflection points (corresponding to the loss of water of hydration and degradation of the organic component) in the case of the biomimetic material prepared with the three-phase method. In the temperature interval between 200 and 400 °C, the MaxResolution analysis (Figure 10B) revealed a single weight loss (ascribable to a sole homogeneous physicochemical structure) in the case of the material prepared with the three-phase method, whereas three evident, distinct weight losses (ascribable to a decidedly more inhomogeneous physicochemical structure consisting of at least three organic entities) were observed in the case of physical blending .

The standard and MaxResolution analyses performed on the bilayer biomimetic materials in toto produced with three-phase method and by physical blending (Figure 11) showed thermal profiles analogous to those obtained for the individual cartilaginous and osseous layers, once again underscoring the fact that, unlike physical blending, the three-phase method makes it possible to obtain more homogeneous, orderly structures characterised by a strong physicochemical interaction and a stable integration between collagen, chitosan and, in the case of the osseous layer, the inorganic component.

Water Uptake Rate test

The uptake capability of the osteocartilaginous biomimetic materials prepared with the pH-dependent three-phase process of synthesis (three-phase method) and by coprecipitation (physical blending) was evaluated in triplicate by means of the water solution uptake rate test, by immersing 4x2x0.8 cm (WxDxH) biomimetic materials in saline aqueous solution (culture medium HANK'S Balanced Salt Solution, containing phenol red) until complete imbibition. In particular, once each sample had been immersed in an upright position inside a beaker containing an adequate volume of HANK'S Solution, a measurement was taken of the time it took for the solution to reach, by capillary action, the upper surface of the material, imbibing it. Figure 12 shows the results of the test performed on two representative preparations: the osteocartilaginous material obtained by means of the pH-dependent three-phase process of synthesis shows a mean water uptake time of 9 seconds, whereas the material obtained by physical blending gave a mean value of 11 seconds, equivalent to 22.2% longer.

The distinctly shorter water uptake time observed for the osteocartilaginous material prepared by means of the three-phase process is directly correlated with the greater homogeneity, structural uniformity, orderliness and spatial organisation thereof compared to the material obtained by physical blending.