WO2007115388A1 - A composite comprising collagen and carbon nanotubes, process of produing same and uses thereof - Google Patents

Abstract

The present invention refers to a process for preparing a biodegradable, biocomposite using collagen and carbon nanotubes. It can be used in the in medical and veterinary areas, for the purpose of regenerating, replacing and reconstituting cells and biologic tissue, either in vitro or in vivo. This composite shows biocompatibility, structural mechanical strength, biodegradability, low cytotoxicity, high hemostasis, capability of inducing and stimulating cell growth and tissue regeneration, besides serving for drugs controlled releasing, local carrying of radiopharmaceuticals and citotoxic substances in several therapeutic applications and procedures, such as cancer therapy.

Description

Title: "A COMPOSITE COMPRISING COLLAGEN AND CARBON NANOTUBES, PROCESS OF PRODUING SAME AND USES THEREOF"

The present invention describes a biodegradable biocomposite material constituted by collagen and carbon nanotubes (CNT), the process for obtaining this material and uses thereof.

Among the uses of that material, uses in the human medical and veterinary areas are pointed out, more specifically in procedures that aim at regenerating, replacing and reconstituting cells or biological tissue either in vitro or in vivo.

These composites have in their composition two or more types of different materials, such as mixtures of metals and polymers, metals and ceramics or polymers and ceramics. The materials on which the composites are produced are usually classified as matrix or reinforcement. The matrix material provides a structure for the composite and fills the empty spaces that remain among the reinforcement materials and keep them in their relative positions. The reinforcement material is that which provides variation in the mechanical, electromagnetic or chemical properties of the composite material as a whole.

As examples, one can cite polymers, which still has a limited use in the biomedical area due to their low biocompatibility, although the use thereof has been increasing enormously in the last few decades. This situation can be changed, since the mixture of carbon nanotubes (CNT) with other materials (such as biocompatible or non-biocompatible polymers) can generate composites with new electrical and mechanical properties.

This type of material can be applied thanks to the development of implantology, which has stimulated researchers to study biomaterials for the purpose of knowing the reactions that occur in the tissue-implant interface. Initially, the implants were carried out with the use of inert materials such as stainless steels and alumina. From the seventies onwards, Per-lngvar Branemark introduced the concept of osteointegration (direct, structural and functional connection between ordered and live bone and the surface of an implant subjected to functional loads). Since then, the researches have been concentrating on materials and projects, with a view to accelerate the osteointegration, with the more specific objective of diminishing the time required for the bone replacement. The biomaterial should enable the sustained growth of the neighboring bone, providing direct contact between the bone and the material, so that the osteointegration phenomenon can take place.

Collagens constitute one of the most abundant families of biomolecules (proteins) in the tissues, and there are at least 20 types of chains that combine with each other to produce different forms of collagen with mechanical and biochemical properties that are differentiated and conformed to the specificity of the tissues.

In the collagen structure more than 10 thousand triple helices of long polypeptides interlace, forming a great diversity of collagen molecules known at present. Collagen is the biomolecule responsible for the integrity and connectivity between the cells of various tissues and organs in the living beings, providing the skin, muscles, tendons, meniscus, ligaments, veins, vessels and arteries, with elasticity and resistance, light transmission in the cornea, distribution of fluids in blood and lymph vessels and in the bone itself. In hard tissues, collagen functions as a support for mineral deposition, constituted chiefly by hydroxyapatite (HAp). In soft tissues, it forms a matrix where various substances and cells are embedded. Collagen acts in maintaining the shape and integrity of the tissues. Collagen is present in: glands, internal organs, blood and lymph vessels, cell walls, bones, skin, ligaments, tendons, cornea, crystalline lens of the eye, cartilages, meniscus, intervertebral disks and blood plasma.

Its molecular structure consists in primary protein sequences, which causes differences between the various types of collagen. Collagen can have helices rich in prolines, glycines, etc. Those amino acids are the important factors in the formation of collagen fibers of superior order structure.

Collagen provides the skin, muscles, tendons, menisci, ligaments, veins, vessels and arteries with elasticity and resistance, light transmission in the cornea, distribution of fluids in blood and lymph vessels in the bone itself, etc. In hard tissues like the bones, collagen functions as a support for mineral deposition. In soft tissues like the skin, collagen forms a matrix where various substances and cells are embedded. It is essential to maintain the shape and integrity of the tissue. In the cardiovascular tissue collagen forms a network through the arteries, giving them a greater expansion power and protecting them from possible injuries by excess expansion. Another important function of collagen is its actuation in aggregating, adhering and activating the platelets. It further acts as a factor of the intrinsic mechanism for the blood to coagulate.

The extraction of collagen and preparation of collagen solution is reported in the literature and can follow numberless procedures as taught in US 6,335,007, US 6,821,530, US 6,197, 934, US 5,263,984, BR 0500456-0, BR 0206520-7, BR 9906101- 5, BR 9404595-0, BR 9405043-0. In a general way, a collagen solution is obtained from animal tendons that are dipped into a 4% acetic acid solution under stirring, and then the solution is centrifuged. The precipitated portion is discarded and the solution is mixed with a saline solution under stirring and again centrifuged. This time, the precipitated portion is reserved and the solution is discarded. The precipitated material must be re- dispersed in 4% acetic acid, this aqueous solution being used for incorporation of the carbon nanotubes.

The aqueous solution of dispersed carbon nanotubes is prepared by adding chemical groups to the carbon nanotubes walls and ends, such a procedure being known as chemical functionalization in an acidic or basic medium. Chemical methods and procedures of functionalizing carbon nanotubes are also widely reported in the literature J. Am. Chem. Soc. 2003, 125, 15174-15182, Chem.Mater. 2005, 17, 3235-3241 , 222, Nano Letters 2002, v. 2, 1, 25-28, J. Phys. Chem. B 2004, 108, 7938- 7943, Chem. Eur. J. 2003, 9, 4000-4008.

The CNTs are metastable forms of carbon resulting from the coiling of carbon atom plane in hybridization sp² with a typical diameter in the order of 1 - 100 nm and a length in the order of 3 - 10 μm. Using a diameter in the order of 1 - 100 nm, quantic effects of confinement make this material to be considered a one-dimensional system and its electronic properties dependent on its crystallography, that is to say, its radius and symmetry (or quirality) can be compared with those of a metal or semiconductor. These nanotubes may be composed of a single- walled nanotubes layer or of multi-walled carbon nanotubes multiple layers. The covalent bond of its atoms provides the carbon nanotubes with characteristics of a material having high strength and its metallic nature provides same with a high thermal and electrical conductivity. In addition, the absence of pending bonds imparts great chemical inertia to this material, which is fundamental for the use thereof in biotechnological and biochemical applications.

The combination of the CNT physical properties with biocompatible polymers opens a field in the direction of a new generation of tissue engineering and enables the use thereof as a support for cell growth and reconstruction of natural tissues subjected to high mechanical tensions, besides enabling alterations in physical properties, such as thermal and electrical conductivity, so as to imitate the chemical and physical conditions existing in the natural biological environment. These nanostructures are used so as to develop new biomterials based on CNT and collagen composites, which aim at modifying, improving and creating new uses in the areas of reconstruction of . tissues and biointegration.

Polymers used in medical applications are grouped in two categories: biostable and biodegradable. The biostable ones are long-life materials, enabling the use thereof in long-lasting implants such as blood vessels, cardiac valves and bone joints. Biodegradable materials are used in temporary implants and usually decompose into smaller molecules, being either metabolized or excreted by the body. Implants using collagen as a host biomaterial can be classified in this second category. Its use is limited due to its low strength. However, with the incorporation of CNT into the collagen, its strength can be greatly improved since such nanostructures have strength 100 times higher than steel and are 1/6 lighter. In addition to the increase in strength, the CNT have a high thermal and electrical conductivity (6000 siemens/cm), flexibility, high specific surface (1000 to 1600 m²/g) and biocompatibility, which make them ideal materials for the creation of biodegradable biocomposites.

The scientific base of bone transplantation was established in the middle ninth century with observations made by OLLIER 91967) on the osteogenic properties of the bone and periosteum, just as by FRIEDLANDER (1985), the beneficial influence of cold in preserving these characteristics. INCLAN₅ in 1982, and WILSON in 1947-51 published studies where they described the use of preserved bone in orthopedic surgery. The need to find a graft material for the segmental bone cracks created on the skeleton has grown day by day, as can be seen in O

the publications of various authors.

The replacements of bone tissues have stimulated, in a general way, researches to find new materials also called biomaterials. Some factors are fundamental in the characteristics of the supports, as for example morphology, dimension, pore distribution and mechanical properties.

Products for tissue engineering as, for example, the porous supports for implants are regulated by the ASTM rules and by governmental bodies of countries like Canada, Japan and USA (LLOYD-EVANS, 2004). Materials intended for use in bone-tissue engineering may be ceramic such as calcium phosphates, bioactive glass, glass-ceramic, alumina and zirconia. They may also be polymeric (either natural or synthetic) like chitosan, (poly) lactic acid (PLA) and (poly) glycolic acid (PGA). The may further be metallic like titanium, stainless steel and still composites.

These materials must have the following characteristics: enabling adhesion and growth of cells; no component or products from its degradation may cause inflammatory or toxicity reactions; they must be three-dimensional in shape; their porosity must provide a large surface area for interaction between the cell and the support; they must have room for surface regeneration of the exracellular matrix and enable diffusion during in vivo culture and adjustable regeneration rate for combining with the rate of regeneration of the tissue of interest. Regulated biomaterials are applied in various situations such as cartilage regeneration based on nanocomposites with carbon nanotubes. Nanoscale composites containing CNT- reinforced PLA have the function of a support for the cartilage like a bone in the field of tissue engineering. A nanocomposite has a unique structure composed by a PLA matrix in the form of nanofibers. This material is characterized by its large surface area in a small portion thereof (1000 m²/g) and by the high porosity produced by electrospinning process with intertwining of the fibers. The porosity of this material is ideal for cell migration and transportation of nutrients, and can be controlled. In vitro studies have shown that the PLA nanofibers containing CNT promote cell growth without visible damages to cell proliferation by the presence of the CNT's. These studies have concluded that a CNT is not a harmful material, being cell friendly.

However, it must be pointed out that the applications of that material are different from the applications for the product obtained according to the present invention. Moreover, since that is a polymer of a material strange to the organism, the risk of rejection is greater.

Studies report various applications of CNT's combined with matrices for biomaterials, including polymers, composites such as epoxy, thermoplastics, gels, polymethylmetacrylate (PMMA), polyacrylonitryl (PAN) and some other polymers, although with different applications. CNT's can also include ceramic matrices like alumina (AI₂O₃), silica (SiO₂) and silicon carbide (SiC) or metallic matrices like (Ni), titanium (Ti) and aluminum (Al) to form composites. The greatest problem in using these composites is the dispersion of CNT's. Actually, several researchers study CNT dispersion and little are known about the interactions that occur during this phenomenon.

At present, biocomposites are being widely used in several areas of veterinary and human medicine and dentistry, as well as in several processes and studies in the biological sciences. This is the case of carbon nanotube composites with diverse polymers such as PMMA, PAM, PVA (polyvinyl alcohol), PGLA, among others. These composites are used for making implantable devices and interventions in these areas, as described in Sakina Sharmin Khan, Carbon Nanotube Based Nanocomposite Fibril for Cartilage Regeneration, Masters of Science in Biomedical Science of Drexel University, September 2002.

In the present case, inventors have developed a composite by using type-l collagen, which is a fibril collagen formed by three (alpha) polypeptide chains. These chains are organized in a triple-helix shape, being called pro-collagen (figure

1)-

The inventors developed a study on the incorporation of the HAp onto the materials, that is to say CNT, collagen and composites, in order to verify whether with a lower concentration of "CA²⁺ ions seeking" phosphate groups it would be possible to obtain responses quite similar to those already found by using HAp, without it being present in the implant material and using the metabolism of the body itself for synthesizing it. Various techniques of evaluating the adequateness of a new material for biomedical applications have been developed in an attempt to simulate the performance of the material after its insertion into the human body. These techniques comprise in vitro and in vivo tests. For in vitro tests, tests for bioactivity in a simulated buffer solution and studies in cell cultures were carried out. These studies comprise tests for cytotoxicity, biochemical measurements of cell activity, evaluation of proliferation, cell growth and morphology, among others.

The biocomposite is formed by mixing two materials of recognized biocompatibility, namel, collagen and carbon nanotubes as described above for the purpose of producing a new biomaterial, which has the following characteristics: structural mechanical strength, biodegradability, low cytotoxicity, high hemostasis, capability of inducing and stimulating cell growth and tissue regeneration, besides serving for drugs controlled releasing, local carrying of radiopharmaceuticals and citotoxic substances in several therapeutic applications and procedures, such as cancer therapy.

This biocomposite is basically a mixture of collagen with carbon nanotubes of single and multiple walls, either functionalized or not, homogeneously dispersed in this collagen matrix, in dry or hydrogelatinous form for use in biodegradable implants, in vitro or in vivo cell growth, cartilage and tendon repair, bone fracture repair, integration of artificial surfaces and biological tissue in general, as well as localized regeneration of bone loss, and further as an agent for transportation of pharmaceuticals with a specific biochemical function, not restricted to these applications.

The biocomposite made of collagen and carbon nanotubes of the present invention is a nanostrucured biomaterial with capability of replacing biological structures and the loss of function of the host tissue, thus enabling the regeneration thereof when it is implanted therein, leading to the restoration and maintenance of the function by means of this replacement.

The main characteristic of this biocomposite is the construction of structures with morphology and porosity controlled by the mixture proportion of its two basic constituents, namely, collagen and carbon nanotubes. This nanostructured material from collagen and carbon nanotubes is simple and inexpensive, easy to manufacture and to handle and is a relatively simple solution for repairing tissues, for use in biodegradable implants and for other interventions in the fields of medicine, orthopedics and dentistry.

This new type of biocomposite is highly porous, with a three-dimensional skeleton, allowing the cells to reach the center thereof, as well as to pass from an end to the other, providing cell reconstitution and close integration in the host matrix. The control of the interlacing between collagen and carbon nanotubes through chemical bonds between these two naterials or by varying the mixture composition further enables the control of the physicochemical properties of the biocomposite. It also permits an additional control of its porosity that is improtant for cell growth, as well as of the velocity of degradation when implanted.

According to the present invention, the collagen used for making this composite is of type I. Type-I collagen is fibrillar, little soluble in an aqueous medium, specifically physiologic solution, and is formed by three polypeptide chains, which are organized in a triple-helix shape, being called procollagen (Figure

1)- This semi-flexible linear macromolecule has length of approximately 300 nm and is 1.5 nm in diameter, its average molecular mass is of 300 KDa and its polypeptide chains are arranged in two equal chains with 1 ,055 amino acid residues and a differentiated one with 1 ,029 amino acids residues, the chains being linked to each other by hydrogen bonds and electrostatic interactions. Its primary structure is characterized by repetition of the triplet Glycine (gly) 33%, Proline (Pro) 12%, Hydroxyproline (Hypro) 11%, the conformation of which generates fibers of great resistance to the tension forces and great elasticity. The present invention also refers to a process of preparing a biocomposite from collagen and carbon nanotubes, so as to create a three-dimensional support for in vitro or in vivo cell growth, where new physicochemical characteristics and properties resulting from this mixture can be controlled by the mixture proportion of these two basic elements, namely collagen in its various arrangement types and carbon nanotubes in its various types.

In one embodiment, the process for preparing the composite of the present invention comprises an electrospinning step, wherein a continuous flow of a PVA: CNT solution is produced by rupture of the surface tension between the liquid and air; the polymeric solution drops come out of the tip of capillary tubes by means of an electric force generated between the source of polymer (capillary tube) and a target (flat Cu plate); the electric field applied induces an alignment of the CNTs parallel to the flow direction; and the alignment of the CNT's is parallel to the axial direction of the fiber. As an alternative for the alignment of the CNTs, the process comprises a sort of spray that expels the polymer mixed with the CNT that melts upon being injected, followed by the dispersion of nanotubes in aqueous solution with polyvinyl alcohol (PVA) or poly(lactic-co-glycolic acid (PLGA), injected with microsyringes in chloroform.

The combination of the physical properties of the carbon nanotubes with those of the biocompatible polymers opens a new filed for a new generation of tissue engineering, enabling the use thereof as a support for cell growth and reconstruction of natural tissues. Among the advantages of the use of the composite of collagen with carbon nanotubes is an increase in the structuring degree and mechanical strength, increase in the thermal and electrical conductivity, giving this material new physicochemical characteristics and properties resulting from this mixture, besides enabling greater control and a desired morphological modification. Figure 2 represents the distribution of rupture stress.

The collagen-carbon nanotube biocomposite is prepared from a solution of collagen and carbon nanotubes dispersed in water or in other organic solvents, with or without addition of surfactants.

The preparation of the collagen consists basically of processes and methodology for extraction, separation and purification of this material obtained from an animal tissue. The extraction should preferably end with aqueous collagen solution with a concentration ranging between 2 and 5 mg/ml. Aqueous collagen in concentrations from 2 to 5 mg/ml enable good incorporation of carbon nanotubes with a dispersion of high degree of homogeneity in the collagen matrix. The aqueous solubilization of this type-l collagen is made by means of acetic acid for breaking the bonds between the collagen helices and later dilution in water. When the collagen is solubilized, its pH must be raised until it reaches 6.5 to 7. In basic aqueous solution, collagen has a tendency to reintegrate the chains, thus inducing the formation of a hydrogel.

It must be emphasized that the dispersion of the carbon nanotubes in solvents is fundamental for promoting a homogeneous distribution of the carbon nanotubes in the aqueous collagen solution, so as to produce a composite with appropriate mixture homogeneity. For producing this composite it is necessary that the amount of carbon nanotubes dispersed in water or in another solvent is previously prepared with a known concentration, and that the aqueous collagen solution is also previously prepared with a known concentration, so as to control the ratio of carbon nanotube to collagen in the manufacture of the composite. The mixing of the two solutions must be aided by mechanical stirring or ultrasonification for 1 and a half hours to 100 hours, so as to guarantee a high degree of homogeneity, depending on the concentration and viscosity of the solutions used in the mixture.

The composite of the present invention is prepared from the two above-described solutions. A solution called "solution 1" consists of a dispersion of collagen fibers in an aqueous medium, prepared by mixing collagen with distilled or de-ionized water for a period of time sufficient to form a homogeneous mixture. The solution pH is adjusted to a value between 2 and 4 by adding acetic acid so as to guarantee good aqueous dispersion of the collagen at a temperature between 4 and 18 ⁰C, thus avoiding its degradation. A solution called "solution 2" consists of an aqueous dispersion of carbon nanotubes with a pH ranging from 2 to 4 at room temperature with a previously known concentration (example 1 ). Solutions 1 and 2 are then slowly mixed under mechanical stirring or aided with ultrasonification. The resulting mixture is called "solution 3". The homogeneity degree is evaluated by simple visual inspection and the mixture is considered homogeneous when carbon agglomerates are not observed. The composite is the result of the homogeneous mixture of the aqueous solution of collagen with carbon nanotubes, until a homogeneous mixture, either aided by mechanical stirring or ultrasonification, is reached. Solution 3 is then subjected to a process of removing the solvent by drying same, which may be aided with vacuum under stirring, at temperatures lower than 6O⁰C and subsequently subjected to a lyophilization step. The process of lyophilizing this composite material consists in freezing with liquid nitrogen, followed by a slow step of removing solvent by vacuum-aided sublimation. The process of preparing the composite may include, prior to the lyophilization step, additional steps of adding active functional agents and biologically active agents, either in the collagen and in the carbon nanotubes. In order to utilize the collagen composite as an implant for replacing, reinforcing or strengthening a body tissue, or for acting as an adhesion barrier, the collagen device is contacted with the body tissue and kept in contact with it until the collagen device has been substantially reabsorbed into the body tissue through surgical procedures and subcutaneous applications.

The exposure of collagen molecules from the open triple helix in an aqueous solution to the dispersed carbon nanotubes induces a non-covalent interlacing or steric coiling of those two materials, significantly increasing the strength and its thermal and electronic properties when compared with the original collagen.

Other forms for binding the collagen and the carbon nanotube may be covalent, via amination of the carbon nanotubes, so as to leave free ends of the NH₂ type (J. Lu et al, Chemical physics Letters 405 (2005) 90-92), which in turn bind to protein ends of the collagen, or through a Van der walls type binding, where there is an interaction between two nanometric structures. Example 1 A composite is prepared by using a predefined concentration of carbon nanotubes and a collagen reference without carbon nanotubes.

By using techniques of electronic scanning microscopy and thermogravimetry, one observes changes in the structural characteristics of the composite, as can be seen in Figure 3, wherein A is collagen without carbon nanotubes and B is collagen with carbon nanotubes. In this figure, an increase in the collagen structure in observed, the increase being greater in the orientation of the collagen fibers, and a decrease in the porosity of the composite. The variation in thermal resistance can be seen through thermogravimetry measurements in figure 4, wherein the dotted black curve refers to collagen without carbon nanotubes and the continuous black curve refers to collagen with carbon nanotubes. In that figure, one observes that the incorporation of carbon nanotubes into the collagen matrix raises the thermal degradation temperature and further exhibits an increase in the strength of about 6x. a) in vitro test

The development of biomaterials that imitate bones has had a strategic approach to the growth of hydroxyapatite (HAp) crystals in physiological conditions. However, some factors affect the mineralization, as for example: the means for controlling this process and the reaction mole ratio, which has primary importance. Inventors have investigated the synthesis of HAp by using a physiologic solution of Ca⁺² and P⁺ ions. The depositions were effected by mixing simultaneously solutions of 5 mM Na₂HPO₄ and 10 mM of CaCI₂ and dipping the materials into this solution.

Before carrying out the mineralization experiments on the nanostructured materials with physiologic concentration, two comparative depositions were made on silicon substrate. In the first one, the substrate was immersed in solution with physiological concentration, using the above-described solutions; and in the second one, it would be immersed ion solution with a high mole concentration (using solutions 5 M of Na₂HPO₄ and M 10 of CaCI₂), always maintaining the proportion between them in the mixture.

In this test, we could observe the process of mineralization on the silicon substrates. The substrate exposed to the solution with a low concentration did not undergo mineralization. In figure 5, one can see a virtually clean substrate, without the presence of HAp crystals. This differentiation of the particulate HAp formation between the two substrates shows that in physiological conditions there is no formation of HAp. Thus, this result indicates that the mineralization on substrates dipped into solution with a physiological concentration should take place with the aid of surface with imperfections or needs chemical groups for binding. In order to prove this argument, a systematic study of the process of mineralization on the nanostructured materials (collagen, composite and CNT) was carried out, following the this procedure: each sample was placed in the physiologic solution of Ca and P. After a week, part of the sample was taken out and analyzed until the maximum time of 6 weeks. Example 2 - Effect of adding nanotubes a) Cellular feasibility in a culture of murine magrophages

The toxicity of CNT was studied by using the technique of evaluating the cellular feasibility by MTT (3~(4,5-dimethylthiazol-

2-yl)-2,5-diphenyl tetrazolium bromide). MTT was used as an indicator of the cellular feasibility through its reduction dependent on the mitochondrial metabolism, generating Formazan crystals inside the cells.

For this assay mice 12 weeks of age were used, which were inoculated with a sodium thioglycolate solution to stimulate the migration of macrophages to the peritoneal cavity, from where they were collected, diluted in a culture medium, centrifuged and re-suspended in a culture medium with concentration of 2 X 10⁶ cells per ml_. Then, these cells were distributed in assay plates with 96 wells and placed in the oven for adhesion of the cells. After a minimum time of 3 hours, the plates were washed twice with a culture medium for removal of dead or non-adhering cells. After that step, culture medium was added to the wells with different concentrations of CNT, part of the wells was filled without the presence of CNT as a form of control. These prepared plates were incubated in a CO₂ oven for 24 and 48 hours; after this period the plates were washed again with culture medium and filled with diluted-MTT solution in a culture medium A, and the plate was incubated again for 4 hours, and during this period the live cells metabolized the MTT, forming Formazan crystals inside them. In order to measure the cellular feasibility, the MTT solution in a culture medium was taken out of the wells and a solution of 10% sodium - dodecylsulfate (SDS) in dimethylformamide and water (1:1) was used to wash the wells, lyze the cells and dissolve the Formazan crystals. The solutions obtained by washing the wells were passed to a new plate and led to an ELISA reader (Emax, Molecular Devices) at 540 nm. This reader verifies comparatively the concentration of Formazan coming from the incubated walls only with the culture media (control) and from the wells that were incubated with CNT. The larger the amount of Formazan, the longer the survival of the cells, that is to say, the greater the cellular feasibility.

Claims

I o

1. A biodegradable, biocomposite characterized by comprising collagen and carbon nanotubes (CNT).

2. A composite according to claim 1 , characterized by comprising a mixture of soluble carbono nanotubes and a collagen solution in any proportions.

3. A composite according to claim 1 or 2, characterized by comprising carbon nanotubes functionalized with any functional group. 4. A composite according to any one of claims 1 to 3, characterized in that the collagen is type-l collagen.

5. A composite according to any one of claims 1 to 4, characterized by comprising a mixture of collagen with single- walled or multi-walled carbon nanotubes, wherein the nanotubes are homogeneously dispersed in this collagen matrix in dry form or in hydrogelatinous form.

6. Process for preparing a biodegradable, biocomposite as defined in claim 1 , characterized in that it comprises adding carbon nanotubes to collagen in order to form a homogeneous mixture thereof.

7. Process according to claim 6, characterized by comprising adding soluble carbono nanotubes to the collagen solution in any proportions.

8. Process according to claim 6 or 7, characterized by comprising adding carbon nanotubes functionalized with any functional group.

9. Process according to any of claims 6 to 8, characterized in that the collagen is type-l collagen.

10. Process according to any of claims 6 to 9, characterized in that carbon nanotubes are added to collagen, a process of adding stability to the composite at a temperature higher than the collagen degradation temperature, due to the »

structuring with carbon nanotubes.

11. Process according to any one of claims 6 to 10, characterized in that the resulting structure construction has morphology and porosity controlled by the mixture proportion of the two basic constituents of the biocomposite.

12. Process according to any one of claims 6 to 10, characterized in that the resulting composite has structural strength, biodegradability, low cytotoxicity, high hemostasis, capability of inducing and stimulating processes of cell growth and tissue regeneration.

13. Process according to any one of claims 6 to 12, characterized in that it comprises an electrospinning step, wherein a continuous flow of a PVA: CNT solution is produced by rupture of the surface tension between the liquid and air; the polymeric solution drops come out of the tip of capillary tubes by means of an electric force generated between the source of polymer (capillary tube) and a target (flat Cu plate); the electric field applied induces an alignment of the CNTs parallel to the flow direction; and the alignment of the CNTs is parallel to the axial direction of the fiber.

14. Process according to claim 12, characterized by comprising a sort of spray that expels the polymer mixed with the CNT that melts upon being injected, followed by the dispersion of nanotubes in aqueous solution with polyvinyl alcohol (PVA) or poly(lactic-co-glycolic acid (PLGA), injected with microsyringes in chloroform.

15. Process according to any one of claims 6 to 14, characterized by the addition of single-walled or multi-walled carbon nanotubes that are homogeneously dispersed in this collagen matrix in dry form or in hydrogelatinous form.

16. Process according to any one of claims 6 to 15, characterized in that the resulting material is a highly porous biocomposite with a three-dimensional skeleton.

17. Process according to any one of claims 6 to 16, characterized in that said collagen is prepared by a method comprising steps of extraction, separation and purification of material obtained from animal tissue.

18. Process according to claim 17, characterized by comprising a step of finishing the extraction in aqueous collagen solution with a concentration ranging from 1 to 5 mg/ml.

19. Process according to claim 18, characterized by comprising aqueous solubilization of type-l collagen by means of acetic acid to break the bonds between the collagen helices and later dilution in water.

20. Process according to any one of claims 6 to 19, characterized by comprising the step of adding a collagen solution to a solution of carbon nanotubes dispersed in water, optionally including surfactants.

21. Process according to to any one of claims 6 to 19, characterized by adding a collagen solution to a solution of carbon nanotubes dispersed in a solution of organic solvents, with the exception of water, optionally including surfactants.

22. Process according to claim 20 or 21 , characterized by the step of obtaining a solution comprising a dispersion of collagen fibers in an aqueous medium, prepared by mixing collagen with distilled or de-ionized water for a period of time sufficient to form a homogeneous mixture.

23. Process according to any one of claims 20 to 22, characterized by the step of adjusting the pH of the solution to a value between 2 and 4 by adding acetic acid.

24. Process according to any one of claims 20 to 23, characterized by preparing a solution comprising an aqueous dispersion of carbon nanotubes with a pH in the range from 2 to 4, at room temperature.

25. Process according to any one of claims 20 to 24, characterized by mixing the solutions of collagen dispersion and nanotubes dispersion slowly under mechanical stirring or stirring aided by ultrasonification. 26. Process according to claim 25, characterized by further comprising the step of removing the solvent of the resulting solution by a drying process at a temperature lower than 60⁰C, optionally aided by vacuum under stirring, and subsequently subjecting the resulting product to lyophilization. 27. Process according to claim 26, characterized in that, prior to the lyophilization step, further including a step of adding functional and/or biologically active agents, to both the collagen and to the carbon nanotubes.

28. Use of a biodegradable, biocomposite as defined in any one of claims 1 to 5 or prepared by a process as defined in any one of claims 6 to 27, characterized in that it is for the repair of cartilages, tendons and bone fractures, the integration of artificial surfaces and biologic tissues, including the localized regeneration of bone losses. 29. Use of a biodegradable, biocomposite as defined in any one of claims 1 to 5 or prepared by a process as defined in any one of claims 6 to 27, characterized in that it is for replacing biologic structures and the loss of function of the host tissue, enabling the regeneration, restoration and maintenance of function.

30. Use of a biodegradable, biocomposite as defined in any one of claims 1 to 5 or prepared by a process as defined in any one of claims 6 to 27, characterized in that it is in medical and veterinary areas, for the purpose of regenerating, replacing and reconstituting cells and biologic tissue, either in vitro or in vivo.

33. Use of a biodegradable, biocomposite as defined in any one of claims 1 to 5 or prepared by a process as defined in any one of claims 6 to 27, characterized in that it is as a system for controlled release of drugs, for local carrying of radiopharmaceuticals and cytotoxic substances.