ES2331678A1 - Three-dimensional macroporous substrates for tissue engineering - Google Patents

Abstract

The present invention describes a three-dimensional substrate that comprises a polymeric matrix and particles, of nanoscale or micron size, of an inorganic material that are dispersed and homogeneously distributed in said matrix. The substrate comprises a continuous network of interconnected macropores and micropores, and may further comprise a continuous biomimetic hydroxyapatite phase. The substrate is useful in tissue engineering applications. The present invention likewise describes a method for producing the substrate and the use thereof.

Description

Three-dimensional macroporous brackets for tissue engineering

Field of the Invention

The present invention fits within the field of tissue engineering, and more particularly refers to a three-dimensional macroporous support, which can be biodegradable and resorbable and comprising an organic polymeric matrix reinforced with inorganic particles dispersed therein, and, optionally, a biomimetic hydroxyapatite coating. The invention also relates to a process for obtaining of said support, as well as its use in applications of tissue engineering

Background of the invention

Massive loss of bone tissue due to different causes such as accidents, serious diseases like cancer etc ... often needs an implant or substitute Bone for recovery.

The most commonly used bone substitute implant is the autograft. However, this technique involves problems such as suffering from pain on the part of the patient, possible necrosis in the area of bone extraction and limitation in the amount of bone material that can be obtained. The use of allografts (corpse bone tissue) has serious drawbacks such as disease transmission or immune rejection [Truumees E, Herkowitz HN. Alternatives to autologous bone Harvest in spine surgery. In: UPOJ volume 12 spring. 1999. p. 77-88. Conrad EU, Gretch DR, Obermeyer KR, et al ; Transmission of the hepatitis-C virus by tissue transplantation. J Bone Joint Surg Am 1995; 77-A: 214-24. Friedlaender GE, Strong DM, Tomford W, Mankin HJ. Longterm follow-up of patients with osteochondral allografts. A correlation between immunologic responses and clinical outcome. Orthop Clin North Am 1999; 30: 583-8].

Faced with these types of strategies each time he charges more important tissue engineering that is defined as the application of engineering principles and methods and life sciences towards fundamental knowledge of relationships between structure and functions in mammalian tissues, normal and pathological; and the development of biological substitutes to recover, maintain or improve tissue functionality [Williams D., Materials Today (2004) 7, 24].

The strategies that follow in tissue and organ engineering usually focus on one of the following three main approaches: cell-based therapies [Ma PX, Materials Today (2004) 7, 30, Rodriguez H., et al , Tissue Engineered Medical Products (Temps). ASM International, STP 1452, PA, 2004, pag. 84], support-based therapies [Langer R., Vacanti JP, Science (1993) 260, 920; Williams D., Materials Today (2004) 7, 24], and therapies based on bioactive molecules [Bloch J., Fine EG, Bouche N., Zurn AD, Aebischer P, Experimental Neurology (2001) 172, 425; King GN et al , Journal of Dental Research (1997) 76, 1460].

The application of the concept of tissue engineering to bone regeneration has allowed the development of different types of three-dimensional macroporous supports, known and also referred to as " scaffolds ", which should serve as support for bone growth (osteoconduction ) and induce the differentiation of osteogenic cells towards osteoblasts and osteoclasts (osteoinduction).

Several materials have proven to be osteoconductors (calcium phosphates, bioactive glasses, ceramics ...). This type of materials allows a direct link to the bone because of the chemical similarity with the mineral components of East. However they do not have good mechanical properties, they are fragile and brittle, which compromises the viability of the implant and much more if the material must be porous. The materials of this type available today, they also do not offer a adequate porosity (neither in proportion nor in size). Also high stiffness of solid or little porous ceramics can bring the appearance of the phenomenon of load protection which is disastrous for the implant and its surroundings.

On the other hand materials have been developed Polymers with mechanical properties closer to bone tissue that you want to restore, "scaffolds", of structure and Morphology similar to bone that you want to proliferate. Though there is a range of bioreabsorbable, biocompatible and application in contact with bone tissue, these materials are not able to establish direct chemical bonds with the bone. This type of approach is described in US2003008395, but the "scaffold" of PLGA disclosed, even if it has a open porous structure, has no features osteoinductors

For the reasons previously stated, in this field lately attention is directed to the materials compounds of a polymeric matrix and inorganic fillers in different proportions It can be ceramic particles bioactive in medium or high proportion (in porous implants up to 60%) in a polymer matrix as the patent shows WO9846164, or a large number of particles with a minimum proportion of organic material as a binder As US4192021 shows. Other patents that propose various composite materials are US5017627 which discloses a non-biodegradable polyolefin material with inorganic particles; US5552454 that discloses waxes and polymeric resins with ceramics biocompatible The mentioned patents consider the use of mentioned materials for obtaining macroporous supports, but they do not describe their synthesis techniques, nor how get the support morphologies that are needed for effective bone regeneration (porosity, pore size, effective surface etc ...).

Most of the inorganic fillers used in the described materials, such as ceramics, calcium phosphates etc., still have a limited bioactivity in nanometric form. A new biomimetic approach has seen the light, inspired by the works of P. Ma, Kokubo et al [Kokubo T, Kushitani H, Sakka S, Kitsugi T, Yamamuro T. Solution able to reproduce in vivo surface-structure change in bioactive glass -ceramic AW. J Biomed Mater Res 1990; 24: 723-34; Oyane A, Kawashita M, Nakanishi K, Kokubo T, Minoda M, Miyamoto T, Nakamura T. Bonelike apatite formation on ethylene-vinyl alcohol copolymer modi.ed with silane coupling agent and calcium silicate solutions. Biomaterials 2003; 24: 1729-35; Oyane A, Kawashita M, Kokubo T, Minoda M, Miyamoto T, Nakamura T. Bonelike apatite formation on ethylene-vinyl alcohol copolymer modified with a silane coupling agent and titania solution. J. Ceram. Soc. Japan 2002; 110: 248-54. Kawashita M, Nakao M, Minoda M, Kim HM, Beppu T, Miyamoto T, Kokubo T, Nakamura T. Apatite-forming ability of carboxyl group-containing polymer gels in a simulated body fluid. Biomaterials 2003; 24: 2477-84]. It involves precipitating minerals developed from a simulated body fluid (SBF) on macroporous supports, as occurs in the human body on different tissues. A technique for coating the supports with biomimetic hydroxyapatite has been developed, which is valid in all materials that have hydroxyl functional groups such as carboxyls, silanoles, TiOH, etc. The published results (Preparation of bonelike apatite composite for tissue engineering scaffold; Hirotaka Maedaa, Toshihiro Kasuga, Masayuki Nogamia, Minoru Ueda, Science and Technology of Advanced Materials 6 (2005) 48-53) allow us to intuit that the biological properties of hydroxyapatite thus obtained are significantly better than those of calcium phosphates or traditional synthetic apatites. The causes that explain these best results are varied. Among others the small size of the crystals, which favors their redisolution in the organism. This is a phenomenon that is not observed with synthetic apatites sintered at high temperatures, which are highly stable and do not decompose. Another cause is that the chemical composition of this type of apatite is much more similar to that of natural bone.

Various are known in the state of the art patent applications, which describe the preparation of "scaffolds" with very diverse characteristics that are obtained through the use of various manufacturing processes and materials for bone regeneration.

Application US2003082808 discloses a polymeric macroporous support with a network of interconnected macropores having a diameter between 0.5-3.5 mm, preferably between 1-2 mm. This support is prepared by a method comprising the combination of selective dissolution and phase inversion techniques, which provides control over the morphology of the formed support; It has utility in the field of tissue engineering, particularly as a support for cell growth in vitro and in vivo . The surface of the support can be modified, for example, by deposition of calcium phosphate particles resorbable by osteoclasts. However, this support has, among others, the disadvantage that its biocompatibility is limited.

Application US2004 / 191292 discloses a material compound, and its use in the field of biomedical engineering, where The composite material comprises bioactive microparticles that They could induce human bone tissue to regenerate. The support uses the combination of silica, calcium, phosphorus microparticles as bioactive substances that could actively induce proliferation and differentiation of human osteoblasts, promoting the formation and calcification of new bone. He support comprises a three-dimensional structure and an external shape  anatomical according to the requirements for regeneration of bone and vascularization. However, the volume of microparticles included have to be very high to have place a sufficient osteoinductive effect on the surface.

Application US2005255159 describes a porous composition comprising a hydroxyapatite (HAp) material, obtained from a mixture of calcium phosphate, calcium oxide and an inorganic porogen that can be removed from the support a once formed. This one is totally inorganic, does not include phase organic, so its mechanical resilience and resistance to fracture are limited, even more so considering the porosities reached. In addition its ability to be biodegraded is also limited

From all of the above, it turns out Obviously, none of the three-dimensional macroporous supports  described in the state of the art, complies with all necessary and desirable requirements for effective application and satisfactory in tissue engineering. Therefore, it still exists the need to provide three-dimensional macroporous media alternatives, which overcome at least part of the disadvantages of state of the art supports.

In this sense, the inventors of the present invention have surprisingly discovered a new three-dimensional support that has properties especially suitable for use in tissue engineering applications. The support has a morphology, characterized by the co-existence of macropores and micropores continuously interconnected with each other as shown in Figure 5 (left). The three-dimensional support is formed by a matrix consisting of an organic polymer and in which particles of a reinforcing material are homogeneously dispersed. The surface of both macropores and micropores can be activated by a suitable procedure, converting the three-dimensional support into a three-dimensional bioactive support. In this sense, said three-dimensional bioactive support is capable of generating, either when implanted " in vivo " or in equivalent conditions " in vitro ", a coating of continuous biomimetic hydroxyapatite that covers all the internal surfaces of the macropores and micropores resulting in a hybrid support with a biomimetic polymer-hydroxyapatite interpenetrated structure as depicted in Figure 5 (right).

The three-dimensional support of the present invention, described in detail below, results especially suitable for use in tissue engineering since allows the growth of cells and their vascularization, essential for the development of functional and three-dimensional tissue. In concrete one of the applications in which it is of great Utility is the manufacture of intervertebral fusion strips.

Brief description of the figures

Figure 1: SEM micrographs of a) a matrix of polycaprolactone without hydroxyapatite coating biomimetics; and b) detail of the micropore structure of the polycaprolactone matrix.

Figure 2: SEM micrographs of a) a matrix of polycaprolactone in which the coating of biomimetic hydroxyapatite; b) of the detail of the structure of the covering; c) and d) hydroxyapatite growth detail biomimetics inside the micropores.

Figure 3: SEM microphoto of the coating continuous biomimetic hydroxyapatite after pyrolizing the polymer matrix.

Figure 4: SEM photomicrograph of a matrix of Nanoparticle reinforced polycaprolactone (mean diameter 100 nm) of hydroxyapatite dispersed in the polymer matrix.

Figure 5: Representation of the macroporous support before (left) and after (right) of its coating with biomimetic hydroxyapatite where (m) indicate micropores and (M) macropores; and where (HAp-m) and (HAp-M) indicate micropores and coated macropores respectively.

Figure 6: Intervertebral fusion strips of bioactive polycaprolactone obtained by the procedure of the invention.

Figure 7: Shows the result of an analysis of EDX mode electron microscopy of a sample coated with biomimetic hydroxyapatite according to the procedure described (support hybrid): shows the constituent atoms of the deposited layer of Hydroxyapatite and chemical composition analysis.

Figure 8: SEM photomicrograph showing a hybrid support (polymer matrix with HAp coating biomimetic) after calcination.

Object of the invention

In one aspect the present invention relates to a new three-dimensional support consisting of a matrix polymeric and particles of nanometric or micrometric size of a inorganic material, dispersed in said matrix and homogeneously Distributed The support has a morphology that presents a network of macropores perfectly interconnected with each other in size between 50 and 1000 microns, and additionally a second micropore network also interconnected with each other, and with macropores, size between 1 and 10 microns. The support has a porosity in volume between 50 and a 95%

In a particular embodiment said support three-dimensional, hereinafter support of the invention, is found activated as detailed in the description. Said activated support It is hereinafter referred to as bioactive support.

In another particular embodiment, the support of the  invention further comprises an inorganic phase consisting of a biomimetic HAp coating, and to which the description in hereinafter referred to as hybrid support.

Another aspect of the invention relates to a new method of obtaining the support of the invention. He The process comprises the steps of: dissolving a polymer in a first solvent, addition of reinforcing particles and their dispersion with ultrasonic techniques in the liquid medium, porogen addition; abrupt solidification of the mixture by cooling, maintaining the homogeneity of the dispersion of the reinforcement material and avoiding its agglomeration; extraction with a second suitable solvent of the first solidified solvent; dissolution and removal of the porogen.

Another additional aspect of the invention is refers to a support obtainable according to the procedure of the invention.

In another aspect the invention relates to a support for use in tissue generation or regeneration.

Therefore, the invention also relates to use of the support of the invention for the generation or tissue regeneration, for example connective tissue, such as bone or cartilage.

In a further aspect the invention is relates to a procedure to generate or regenerate tissue that comprises obtaining a bioactive support or a hybrid support according to the invention, and culturing said tissue cells in said support selected. Optionally the procedure can include also planting with support cells.

Description of the invention

The object of the present invention relates to a three-dimensional support consisting of a polymeric matrix and reinforcing particles dispersed in said matrix, of size nanometric or micrometric of a suitable inorganic material, and homogeneously distributed in the matrix (Figure 4).

The advantage of this support, which does especially effective in tissue applications, resides in the morphology of the same since it presents a macropore network perfectly interconnected with each other of sizes included between 50 and 1000 microns, and additionally a second network of micropores also interconnected with each other, and with macropores, of size between 1 and 10 microns (Figure 1). The support it has a porosity in volume between 50 and 95% and a high specific surface This morphology confers advantages to the new support, and allows the activation of the surfaces of the micropores and macropores, which facilitates and accelerates the generation of a  biomimetic HAp coating as described below. The Figure 1 shows the double porous structure of a matrix PLC polymer with micropores and micropores interconnected between yes. The SEM scanning electron microscopy image (Figure 1) corresponds to a cryogenic fracture of a matrix of polycaprolactone The micropores are clearly seen in the fracture section You can also clearly see the perfect interconnection between macropores with connecting throats of average diameter sizes greater than 100 µm. The structure morphology both in regards to the fraction in volume of macropores, micropores, their sizes or the size of the throats of connection between pores can be controlled during method of obtaining varying the chemical composition and procedure parameters

The polymer that is used in the present invention can be biodegradable. The polymeric matrix may comprise any conventional polymer, preferably biocompatible, and reabsorbable, in particular, when the matrix is intended for physiological applications. The term "biocompatible" refers in the present invention to being non-toxic and allowing cells to colonize it. The term "resorbable" in the present invention refers to the support disappearing over time when it is inside an animal body as it is replaced by regenerated tissue. The polymer must be selected so that it can be activated superficially by the creation of crystallization nuclei, inducers of the subsequent crystallization of biomimetic hydroxyapatite. That is, the polymer must comprise hydroxyl groups, such as carboxyl groups, silanoles, TiO2 among others and mixtures thereof. In the context of the present invention "biomimetic hydroxyapatite" refers to hydroxyapatite that is deposited naturally " in vivo " or that is deposited by immersing the support in an equivalent solution that reproduces the conditions in vivo , of the same salt composition as human plasma (Simulated Body Fluid or SBF) for the necessary time. Among such polymers and for illustrative purposes only may be mentioned, among others, polycaprolactone (PCL), polylactic acid (PLA), lactic acid and glycolic acid (PLGA) copolymers, lactic acid and ethylene glycol (PLLA-co-PEG) copolymers, lactic acid lysine copolymers, (PPF), ethylene glycol and fumaric acid copolymers, polypropylene fumarate, as well as mixtures thereof. In a preferred embodiment the polymer matrix comprises a polymer approved for clinical use, such as polycaprolactone (PCL).

The polymeric matrix comprises particles of a suitable inorganic material or reinforcement material, which may be, among others, hydroxyapatite, fluorohydroxyapatite, calcium disilicide, calcium phosphate, and mixtures thereof. In general, the size of said particles is between 50-300 nm. In a preferred embodiment, the particles have a size of less than 100 nm so that their effect as mechanical support of the support and its biodegradability are greater. Some nanoparticles can be observed on the surface of the polymer matrix before being coated with HAp (Figure 4). The characteristics of the three-dimensional support of the invention vary depending on its chemical composition; they have porosities between 50-95%, preferably equal to or greater than 70%; elastic modules between 0.1 MPa and 100 MPa, and in vitro degradation rates between 4 and 12 months. The characteristics of the support of the invention can be varied in a simple manner by a person skilled in the art within the ranges described to adapt to the needs required by the specific application.

In a particular embodiment the support of the Invention is a bioactive support. The bioactive support is obtained as explained below in the description, and refers to a support according to the invention in which the micropore surfaces and macropores have crystallization nuclei, inducers of hydroxyapatite crystallization, preferably HAp biomimetics

In the context of the present invention the term "bioactive" refers to the support, refers to the ability of the support to induce the proliferation and differentiation of osteoblasts, to induce the formation and calcification of new bone, and the ability to restore physiological function at the molecular and cellular level. This bioactive ability can be demonstrated if the support is implanted in vivo , or alternatively as in the present invention, by applying the conventional method accepted in this field of the technique, which consists of immersing the bioactive support in a simulated body fluid solution, which It simulates blood plasma and observe how the growth of a biomimetic HAp coating takes place.

In another particular embodiment the support of the invention is a hybrid support, constituted by the support three-dimensional previously described, and a continuous coating and homogeneous biomimetic hydroxyapatite on the surface of macropores and micropores. Adhesion to the polymer matrix of the Biomimetic hydroxyapatite is very high and manages to confer Support good mechanical properties and properties of osseointegration and osteoinduction. The structure of the support hybrid, seen in Figures 2a and 2b in which it is shown clearly that the polymer matrix, and the hydroxyapatite phase biomimetics intermingle at the microscopic level and so highly interpenetrated Both the polymer matrix and the Hydroxyapatite constitute continuous phases. This continuity, is shows in Figure 3 which shows a photomicrograph SEM of a hybrid support after having pyrolized the matrix polymeric, leaving only the biomimetic HAp phase.

The new support of the invention can be molded in the desired size and geometry, depending on the application to the one that is destined; It is biocompatible, osteoinductor, osteoconductor, and preferably it is biodegradable and bioabsorbable. Because of his high specific surface area, and its morphology, bioactivity is also very high which constitutes an important advantage, for example, in the field of bone regeneration. High surface specific is also associated with high reactivity of support, and mineral dissolution / precipitation processes which constitute the biomimetic HAp are favored and accelerated. In addition, these phenomena are linked to the precipitation of organic substances of importance for the differentiation of osteogenic cells, such as various proteins and cytokines. The morphology is especially advantageous since in addition to the high specific surface due to microporosity, presence and Macropore size allows the entry of cells, and the tissue vascularization.

In another aspect the invention provides a procedure for obtaining a support according to the invention which It comprises the stages of:

(i) contacting a solution in a mold of a polymer in a first solvent and size particles nanometric or micrometric of an inorganic material and shake the ultrasonic mixing;

(ii) add porogen particles of a size between 50 and 1000 µm and homogenize the mixture resulting;

(iii) freeze the homogeneous mixture until obtaining a solidified piece;

(iv) cold extract the first solvent from the piece solidified by dissolving it in a second solvent;

(v) extract at room temperature the Porogen particles dipping the piece in a third solvent  which dissolves said porogen particles;

(vi) obtain a polymeric support with reinforcing particles

In stages (i) and (ii) a dissolution of a polymer in a concentration generally comprised between 10% to 40% by weight of polymer with respect to total weight of the solution. This percentage by weight varies according to the type of polymer and solvent used. The solvent can be in principle any suitable organic solvent capable of dissolve the polymer such as dimethyl sulfoxide (DMSO), methylene chloride, ethyl acetate, chloroform, n-heptane, n-hexane, n-pentane, dioxane, benzene, xylene, acetone, Naphthalene and its mixtures. In a particular embodiment the solvent is dioxane.

The porogenic material can be monosaccharides or polysaccharides such as glucose, sugar, organic salts, inorganic salts, acrylic microspheres, polymeric microfibers in the proportion and the appropriate size according to the macroporosity that is to be obtained. Typically said proportion is between 40/60 to 70/30 by weight of porogen particles with respect to the weight of solution. The diameter size of the porogen particles is generally between 50 and 1000 microns, preferably between 100 and 300 µm. The reinforcing particles are first added to the polymer solution, which is homogenized with ultrasound before mixing with the porogen particles; This prevents them from agglomerating or accumulating in certain parts of the support of the invention.
tion.

The homogeneous mixture consisting of a polymer in a first solvent, the reinforcing particles and the Porogen particles are introduced into a mold, with the shape and dimensions of the support piece to be obtained. Likewise, for non-complex scaffold forms can be used after obtaining, various cutting tools to obtain the desired geometry. For example in figure 6 a strip is shown of intervertebral fusion generated according to the procedure of the invention.

The mold has to withstand the solvent organic and sudden temperature changes during the procedure, so it has to be stable chemical and thermally Generally a resin mold is used, of polytetrafluoroethylene (PTFE) Teflon or metal. In general it closes the mold, and the homogeneous mixture in the closed mold is subjected to the next stage

In step (iii) the homogeneous mixture is frozen so that it stabilizes, until a solidified piece is obtained. The freezing can be done conventionally for example by immersing the closed mold with the mixture in a container with liquid nitrogen or simply by its introduction in a device at a temperature below the solidification of the First solvent used. Freezing temperature varies depending on the nature of the first solvent used with the polymer. The solidified piece is demoulded next quickly to later extract the first solvent with a second cold solvent.

In step (iv) the first solvent is removed of the cold solidified piece by dipping it in a second solvent that is at a temperature, than in a particular embodiment is -20 ° C. The extraction takes place by cold diffusion, so that the temperature of the first solvent do not exceed that of its melting point and it passes directly from the crystallized state, to be dissolved in said second solvent. This second solvent may in principle be any solvent. Organic, inorganic and their conventional mixtures. The selection of the same can easily be done by the person skilled in the art, in function of the first solvent. During the extraction of the first solvent, the second solvent can be renewed several times immersing the piece in fresh solvent to reduce the time necessary for the first solvent to diffuse completely out of the solidified piece. The extraction takes place during the time necessary to allow the dissemination of the entire first polymer matrix solvent. In a particular embodiment said solvent is selected from ethanol and mixtures of ethanol-water. It is also possible to make a solvent extraction from the support at least partially by sublimation thanks to the application of pressures closest to the possible vacuum.

Then, in step (v), the piece solidified is brought to room temperature, and particles of Porogen are extracted by immersing the piece in a third solvent capable of dissolving said porogen particles. Said third Solvent can be renewed to completely extract the porogen. In a particular embodiment said third solvent may be, among others, ethanol, water, acetone, and mixtures thereof. Matrix polymer resulting from this stage is washed and dried to obtain the support of the invention (step (vi)).

In a particular embodiment of the procedure of the invention, the method further comprises a step of surface activation treatment of the support, in which they form nuclei distributed by the macropore surfaces and of micropores. These cores are inducers of subsequent crystallization of biomimetic HAp. The support obtained is a bioactive support.

As proof that activation of the support surface takes place and leads to the formation of crystallization inducing nuclei in SBF, the bioactive support is introduced again in SBF and the biomimetic hydroxyapatite is allowed to crystallize for a time between a few days to two weeks The longer the crystallization time, the greater the thickness of the deposited biomimetic hydroxyapatite coating layer. In the simulated body fluid (equivalent and that reproduces the conditions in vivo in which the bioactive support is implanted) a biomimetic hydroxyapatite layer grows. In this sense, a simulated body fluid is used with the composition of blood plasma, described by Kokubo (Biomaterials 27 (2006) 2907-2915- How useful is SBF in predicting in vivo bone bioactivity? Tadashi Kokubo, Hiroaki Takadama). Figure 2 shows SEM photomicrographs of the inner surface of the coated support. This phase of biomimetic hydroxyapatite is so continuous and effective (Figure 2c and 2d) that when the polymer matrix is completely eliminated by pyrolysis, a treatment that does not affect the biomimetic hydroxyapatite phase, its structure remains unchanged and continuous (Figure 3 ). This is due to the penetration of the biomimetic hydroxyapatite coating in the micropores, which gives continuity to the biomimetic hydroxyapatite phase and the anchor firmly to the polymer matrix (Figures 2c and 2d).

The treatment to activate the surface of the macro and micropores, to optionally carry out a subsequent crystallization of biomimetic hydroxyapatite either in vivo or in vitro , comprises the following steps:

one)

exposure support treatment to a plasma of a gas or by immersion in a solution of sodium hydroxide.

2)

immersion the support obtained in 1 alternatively in solutions containing respectively Ca 2+ or PO 4 3-.

3)

immersion of the support obtained in 2) in simulated body fluid (SBF).

       \ vskip1.000000 \ baselineskip
    

Stage 1) introduces on the surface of the macro- and micropores functional groups containing groups hydroxyl, such as carboxyl, silanol, titanium hydroxide, between others, and their mixtures. The gas can be among others, argon, nitrogen or oxygen Then in stage 2) they are generated nuclei of calcium phosphate on the surface of micropores and support macropores characterized by their high surface area specific, by alternating immersion of the support obtained in 1) in solutions containing respectively Ca 2+ or PO 4 3-, for example calcium chloride and potassium phosphate. In step 3) the support obtained in 2) is immersed in a fluid simulated body (SBF) for a period typically less than 24 hours to create the hydroxyapatite inducing nuclei in order to that the crystallization of biomimetic hydroxyapatite once the support has been implanted

       \ newpage
    

In a preferred embodiment the following composition of the SBF buffered with hydrochloric acid e tris (hydroxymethyl) aminomethane hydrochloride (solution from modified Kokubo):

one

       \ vskip1.000000 \ baselineskip
    

In another particular embodiment, the procedure of the invention further comprises a step of coating the internal surfaces of the macro- and micropores, of the support, or of the bioactive support in a simulated body fluid (SBF). The support submerges for a period typically between 24 hours and 2 weeks Depending on the weather, a coating of hydroxyapatite of variable thickness (the longer the longer deposition) that completely covers the macro surface and micropores A hybrid support with a structure is thus obtained interpenetrated as described and illustrated in the invention.

The bioactive or hybrid supports of the Invention are suitable for application in tissue engineering. In one aspect the invention therefore provides a support for its use in the generation or regeneration of tissue.

In addition the inventors have observed that after of two weeks in concentrated SBF (SBFx2), a consistent mineral deposition, which represents for all samples tested, both pure polymer scaffolds and polymers reinforced with hydroxyapatite microparticles, a weight gain of at least 25%. It is noteworthy that the high specific surface of the samples, due to microporosity, It allows an optimal deposition of minerals.

Also, in another aspect the invention relates to the use of the support of the invention in tissue generation or regeneration. In a particular embodiment, a bioactive or hybrid support is used. The generation or regeneration of tissue includes bone repair or replacement, intervertebral fusion strips, implantation of bone tissue prosthesis in vivo , among others.

The tissue can be for example connective tissue, such as bone or cartilage. Bioactive supports and hybrid supports are used in vivo or in vitro .

In a further aspect the invention is It therefore relates to a procedure to generate or regenerate fabric comprising:

provide a bioactive support or a support hybrid according to the present invention, and

culturing cells of a selected tissue in vitro on said support,

and optionally implant the support obtained in the previous stage in a human or animal.

Optionally the procedure can include also a planting with support cells prior to its cultivation and implant.

Differentiated cells can be used as osteoblasts, as well as pluripotential cells such as those of the bone marrow in conjunction with chemical signals such as the factor of GMP1 bone growth.

Also the present invention relates to a method of treatment for the generation or regeneration of a tissue in a subject comprising implanting a bioactive support or a hybrid support in said subject in need thereof, where subject is an animal including the human being. As mentioned above, the support may have been treated prior to its implantation by in vitro cultivating the support with the cells of interest, and / or sowing it with said cells.

Below are examples illustrative of the invention set forth for a better understanding of the invention and in no case should be considered a Limitation of its scope.

Examples Example 1 Procedure for obtaining a bioactive reinforced support according to the invention 1. Preparation of the support (scaffold)

A 20% solution by weight of polycaprolactone (PCL) in dioxane. 10% was then added by weight (with respect to PCL weight) of 100 nm HAp particles in diameter to the PCL solution, which were dispersed by Ultrasound exposure for half an hour.

Subsequently the previous solution was mixed with sucrose in 5/6 weight ratio (solution / sugar), after sifting the sugar to obtain a size of particle between 200-300 microns. Mix resulting was placed in a Teflon mold that was dipped in liquid nitrogen for five minutes. He then poured cold ethanol at a temperature of -10 ° C in the mold and the mold is placed in the freezer at a temperature of -10 ° C. He changed the ethanol three times and the piece was turned around to spread all dioxane out of it. After two days, the freezer piece, cleaned with ethanol and put in water to dissolve the sugar, at room temperature. He changed the water three times, and after three days it was removed and dried.

2. Preparation of activated support

The support was immersed in a 1 molar solution of soda for a day. To ensure that the soda penetrates well into  all micropores of the support, the structure was subjected to successive depressurization and pressurization cycles until no more bubbles came out of the stand. It was left one day at temperature ambient and washed with distilled water.

The structure immediately submerged during ten seconds in calcium chloride concentration 0.05 mol.L -1, and rinsed with distilled water. Then you dipped for ten seconds in potassium phosphate 0.05 mol.L -1, and rinsed with distilled water. This cycle was repeated. five times. Then the structure was placed in a jar of glass with simulated body fluid (Simulated Body Fluid or SBF; composition and concentrations indicated above in the description of the invention). The hydroxyapatite was allowed to crystallize during two hours. It was then washed with distilled water and dried.

In this way the activated support was obtained which It is the basis for manufacturing any type of scaffold hybrid. The geometry of the material obtained depends on the mold selected.

       \ vskip1.000000 \ baselineskip
    

Example 2 Procedure for obtaining and studying the properties of a scaffold hybrid

Several supports were prepared according to invention, varying the proportions of PCL in dioxane between 10 at 20% by weight. In addition, the solution was mixed with several Porogen / dissolution ratios (between 4/5 to 5/4).

The samples (supports) obtained were analyzed to see the influence of the parameters indicated in the porosity resulting from the support. The porogen used is polyethyl methacrylate (PEMA).

It has been proven that the concentration of dissolution influences the quantity and morphology of the micropores, while the amount of porogen influences the amount of macropores and the interconnection between them. Mechanical values they are very sensitive to the increase in macroporosity, obtaining Very soft samples if the porosity is high.

The following table describes the different support samples that have been manufactured and the porosity in% of volume obtained:

2

To the different manufactured samples of supports, the surface activation procedure was applied to them, and to simulate the scaffold situation once implanted, it let biomimetic hydroxyapatite crystallize for two weeks, changing the SBF a week to replace the loss of Ionic species in the environment due to deposition.

An elementary analysis of the surface of the scaffolds using electron microscopy in EDX mode, in which the x-rays emitted by the sample are analyzed during de-excitation after the collision with the electrons Primary These x-rays are characteristic of atoms constitutive of the sample and accurately analyze the composition Chemistry of the deposited layer of hydroxyapatite. The results obtained in all samples with different composition and time Immersion in SBF show, as seen in Figure 7 impurities such as magnesium, sodium, and chlorides, which have crystallized next to calcium phosphate; that is typically the case in the bone hydroxyapatite, while hydroxyapatite synthesized in laboratory and sintered is usually pure, highly crystallized, which hinders its resorption once implanted.

In SEM photomicrograph (Figure 8) you can observe a polycaprolactone coated scaffold of biomimetic hydroxyapatite, following the procedure of activation explained above and submerged in SBF 3 weeks and subsequently calcined by heating to a temperature of 1000 ° C It is observed that after calcining this sample the residue maintains its physical integrity, proof that the two phases are highly interpenetrated and interconnected.

Claims (37)

         \ global \ parskip0.970000 \ baselineskip
      

1. A three-dimensional support consisting of a polymer matrix comprising particles of nanometric size or micrometric of an inorganic material dispersed and homogeneously distributed in said polymer matrix. 2. Support according to claim 1, which It has a double porous structure with a macropore network interconnected with each other in size between 50 and 1000 microns with connecting throats between the macropores of sizes of diameter means greater than 100 µm, and a second network of micropores interconnected with each other, and with macropores, of size between 1 and 10 microns. 3. Support according to claim 1 or 2, which it has a porosity in volume between 50 and 95% and an elastic module comprised between 0.1 MPa and 100 MPa. 4. Support according to any of the claims 1 to 3, wherein the matrix polymer Polymeric is biocompatible. 5. Support according to any of the claims 1 to 4, wherein the polymer comprises groups hydroxyl 6. Support according to any of claims 4 to 5, wherein the polymer is selected from polycaprolactone, polylactic acid, lactic acid and glycolic acid copolymers, lactic acid and ethylene glycol copolymers, co-
polymers of lysine and lactic acid, copolymers of ethylene glycol and fumaric acid, polypropylene fumarate and mixtures thereof. 7. Support according to any of the claims 4 to 6, wherein the polymer is polycaprolactone 8. Support according to any of the claims 1 to 7 wherein the size particles nanometric or micrometric are hydroxyapatite, fluorohydroxyapatite, calcium disilicide, calcium phosphate and its mixtures 9. Support according to claim 8, wherein the particles have a size between 50-300 nm. 10. Support according to claim 9, wherein The particles have a size smaller than 100 nm. 11. Support according to any of the claims 1 to 10 further comprising on the surfaces of micropores and macropores crystallization inducing nuclei of hydroxyapatite. 12. Support according to any of the claims 1 to 10, further comprising the matrix polymeric a continuous and homogeneous hydroxyapatite coating biomimetics on the surface of macropores and micropores. 13. A procedure to obtain support according to any of the preceding claims comprising the stages of:

(i)

contacting in a mold a solution of a polymer in a first solvent and size reinforcing particles nanometric or micrometric of an inorganic material and shake the ultrasonic mixing;

(ii)

add porogen particles of one size between 50 and 1000 pm and homogenize the mixture resulting;

(iii)

freeze the homogeneous mixture until you get a solidified piece;

(iv)

cold extract the first solvent from the piece solidified by dissolving it in a second solvent;

(v)

extract at room temperature the particles of porogen immersing the piece in a third solvent that dissolves said porogen particles;

(saw)

obtain a polymeric support with particles of reinforcement.

14. Method according to claim 13, in which dissolution of a polymer has a concentration comprised between 10% to 40% by weight of polymer with respect to total weight of the solution. 15. Method according to claim 14, in where the solvent is selected from dimethylsulfoxide, methylene chloride, ethyl acetate, chloroform, n-heptane, n-hexane, n-pentane, dioxane, benzene, xylene, acetone, Naphthalene and its mixtures. 16. Method according to claim 15, in which the solvent is dioxane. 17. Procedure according to any of the claim 14 or 15, wherein the polymer solution is mixed with the reinforcing particles and homogenized with ultrasound

         \ global \ parskip1.000000 \ baselineskip
      

18. Method according to claim 13, in which the material of the porogen particles is selected from between the group formed by monosaccharides, polysaccharides, salts organic, inorganic salts, acrylic microspheres, microfibers Polymers and their mixtures. 19. Method according to claim 18, in which the porogen particles are added to the solution of polymer with the reinforcing particles in a proportion between 40/60 to 70/30 by weight of porogen particles with respect to the weight of the solution. 20. Method according to claim 18 or 19, in which the size of the diameter of the porogen particles It is between 100 and 300 µm. 21. Method according to claim 13, in which the homogeneous mixture is frozen until obtaining a piece solidified 22. Method according to claim 13, in which the first solvent is removed from the solidified part by cold diffusion by immersing it in a second solvent that found at a temperature of -20ºC and without the temperature of First solvent exceeds that of its melting point. 23. Method according to claim 22, in that the second solvent is at a temperature of -20 ° C. 24. Method according to claim 23, in which the second solvent is selected from a solvent Organic, an inorganic and its mixtures. 25. Method according to claim 24, in which the second solvent is selected from ethanol and mixtures  of ethanol and water. 26. Method according to claim 13, in that the porogen particles are extracted at room temperature of the solidified piece submerging the piece in a third solvent capable of dissolving the porogen particles. 27. Method according to claim 26, in which the third solvent is selected from ethanol, water, Acetone and its mixtures. 28. Procedure according to any of the claims 13 to 27, further comprising a step of surface treatment of the support, which forms inductive nuclei of the crystallization of distributed biomimetic hydroxyapatite on the surfaces of macropores and micropores. 29. Method according to claim 28, in which the treatment stage comprises the following stages:

one)

exposure support treatment to a plasma of a gas or by immersion in a solution of sodium hydroxide.

2)

immersion the support obtained in 1) alternatively in solutions that each contain ions Ca 2+ or PO 4 3-.

3)

immersion of the support obtained in simulated body fluid, (SBF).

30. Method according to claim 29, in which the simulated body fluid presents the following composition capped with hydrochloric acid and tris hydrochloride (hydroxymethyl) aminomethane (Kokubo solution modified): 3 31. Procedure according to any of the claims 13 to 30, further comprising a step of coating of the macropore's internal surfaces and micropores, comprising dipping a support or a support bioactive in a simulated body fluid to obtain a support hybrid. 32. Support obtainable according to any of the claims 13 to 31. 33. Support according to any of the claims 1 to 12 for use in the generation or regeneration of tissue. 34. Use of the support according to any of claims 1 to 12, for the generation or regeneration of tissue in vitro . 35. Use according to claim 34, wherein the tissue is connective in vitro . 36. Use according to claims 34 or 35 wherein the support is bioactive or hybrid in vitro . 37. Procedure to generate or regenerate fabric comprising: get a bioactive support or a support hybrid, and culturing tissue cells in vitro on said support, and optionally seeding with support cells prior to cultivation