WO2009101228A1

WO2009101228A1 - Co-continuous hybrid structure for the regeneration of bone defects

Info

Publication number: WO2009101228A1
Application number: PCT/ES2009/000081
Authority: WO
Inventors: José Luís GÓMEZ RIBELLES; Manuel MONLEÓN PRADAS; Julio José SUAY ANTÓN; Myriam Lebourgh
Original assignee: Universidad Politécnica De Valencia; Fundación Comunidad Valenciana - Centro De Investigación Principe Felipe
Priority date: 2008-02-14
Filing date: 2009-02-12
Publication date: 2009-08-20
Also published as: ES2330823A1; ES2330823B2

Abstract

The invention relates to a method for obtaining a scaffold consisting of a porous PCL matrix and a hydroxyapatite coating, comprising the following steps: (i) preparing a homogeneous PCL solution in a mixture of two solvents, namely a PCL solvent and a PCL nonsolvent; (ii) filling a mould with the homogeneous solution; (iii) cooling the homogeneous solution to a temperature lower than the temperature at which the separation of the two liquid phases occurs; (iv) isothermal treatment; (v) solidifying both phases, reducing the temperature; (vi) extracting the PCL solvent and the PCL nonsolvent, together with a third solvent; and (vii) depositing biomimetic HAp. The structure is suitable for use in tissue engineering.

Description

HYBRID CO-CONTINUOUS STRUCTURE FOR THE REGENERATION OF

BONE DEFECTS

FIELD OF THE INVENTION

The present invention falls within the field of tissue engineering, and more particularly it relates to a three-dimensional macroporous support, comprising a porous polycaprolactone matrix and a biomimetic hydroxyapatite coating. The invention also relates to the method of obtaining said support, as well as the use thereof in applications such as bone tissue regeneration.

BACKGROUND OF THE INVENTION

Due to the suffering of cancers, accidents or bone degeneration a massive loss of bone tissue can occur, and lead to the need for a bone graft. An ideal bone graft to repair bone defects should allow bone cells to grow in the affected area, to restore the function and physical integrity of the bone. Today, autografts are the preferred option for bone repair because they are biocompatible and there is no risk of disease transmission. However, the limitations of autografts are the small amount of tissue obtainable, the double operation necessary to carry out the graft, and the morbidity at the extraction site, usually associated with discomfort and suffering for the patient. Allografts, which consist of grafting the tissue extracted from the bone of a corpse, are also solutions but are increasingly ruled out due to the risk of disease transmission and immune rejection. To solve the problems associated with grafts Bone, many researchers have tried to develop artificial substances that can serve as bone graft. In the state of the art the qualities that such material must possess to be successfully implanted are defined. First, the material must be biodegradable so that as the new bone is formed it can grow at the graft site, since it disappears over time. Both the material and its degradation products must be biocompatible, so as not to present any risk to the patient's health. Second, the material must maintain mechanical characteristics similar to those of the bone, so that the loads transmitted in the implant favor bone remodeling and calcification. Third, the material must be osteoconductive, that is, it must provide the cells with a support to adhere and proliferate until the entire implant is colonized. Osteoconductive materials include calcium phosphate-based ceramics, such as hydroxyapatite (natural inorganic component of bone), calcium triphosphate or bioglass (Bioglass ®).

The ceramics and bioglasses are compatible because of the similarity of their chemical structure with that of the native bone, however they are difficult to process, especially in porous form, and they do not have the flexibility that gives the bone its organic part, the collagen, being very rigid and brittle. In addition, sintered hydroxyapatites are highly crystalline and do not degrade easily, which in the long term can prevent complete bone regeneration.

Biodegradable polymers that arouse much interest in the field of tissue engineering are for example polycaprolactone (PCL), polylactic acid (PLA), the polyglycolic acid (PGA), acid (lactic-co-glycolic), etc. They are easy to shape and transform, and degrade at a rate that can be adapted to the bone regeneration rate. Although scaffolds can be synthesized from them in a simple way and have good mechanical properties, pure polymers are not ideal for this use since they are not very osteoconductive. The neoformed bone will not adhere or grow well in pure materials. Due to this fact, logically, the attention of the new investigations carried out has turned to composite materials (which, like bone, which combines an organic phase, collagen, with an inorganic phase, hydroxyapatite), contain both polymer and ceramic. To achieve the composite material, specific inorganic particles of reduced size are incorporated into a matrix of the selected polymer. In addition to an improvement of mechanical properties due to the reinforcing effect of said particles, the bioactivity and osteoconductivity of the support is expected to grow. Several studies have demonstrated the feasibility of this approach

(Biomaterials, in press; Fabrication of three-dimensional polycaprolactone / hydroxyapatite tissue scaffolds and osteoblast-scaffoid interactions in Vitro, Lauren Shor, Selcuk Guceri, Xuejun Wen, Milind Gandhi, Wei Sun). However, the amount of particles that appear on the surface is quite small and the osteoinductive effect is consequently also so. That is why a very high amount of load is necessary for this effect to be significant.

A procedure proposed by Kokubo et al. [Biomaterials 27 (2006) 2907-2915- How useful is SBF in predicting in vivo bone bioactivity? Tadashi Kokubo, Hiroaki Takadama] to quantify the osteoinductivity of a support is immersion in a simulated body fluid (aqueous solution with the same amount of electrolytes as the blood plasma). An osteoinductivity test is to have achieved the deposition on the surface of the material of an inorganic phase based on calcium phosphate, similar to the hydroxyapatite of the bone.

In many studies, procedures are proposed so that biomimetic hydroxyapatite can be nuclear on the surface of the polymeric material in order to increase cell adhesion and bioactivity. The presence of biomimetic hydroxyapatite in the graft can favor bone remodeling by redisolving the deposited apatite. Additionally and due to a greater excess of ions in the medium, the presence of hydroxyapatite may serve to regulate the pH of the medium around the graft since some degradation products of the polymers are acidic (for example in the case of polylactic acid) . Techniques for coating biomimetic hydroxyapatite different polymeric materials have been developed as long as they have hydroxyl functional groups such as carboxyls, silanoles, TiOH, etc. The published results [Preparation of bonelike apatite composite for tissue engineering scaffold; Hirotaka Maeda, 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 such apatite are significantly better than those of calcium phosphates or traditional synthetic apatites. The causes that explain these best results are varied. Among others it stands out that the average crystal size of Apatite formed by this procedure is small, which favors the redisolution of crystals in the body. 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.

Patent applications are known in the state of the art, which describe the preparation of "scaffolds" with very different characteristics that are obtained by using 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 0.5-3.5mm, preferably between 1-2mm. This support is prepared by a method comprising the combination of the techniques of selective dissolution and phase inversion, which provides control over the morphology of the formed support, has utility in the field of tissue engineering, particularly as a support for cell growth in In vitro and in vivo. The surface of the support can be modified for example by deposition of resorbable calcium phosphate particles by osteoclasts. However, this support has, among others, the disadvantage that its biocompatibility is limited.

Application US2004 / 191292 discloses a composite material, and its use in the field of biomedical engineering, which comprises bioactive microparticles that could induce human bone tissue to regenerate. The support uses the combination of microparticles of silica, calcium, phosphorus as bioactive substances that could actively induce the proliferation and differentiation of human osteoblasts, promoting the formation and calcification of new bone. However, the volume of microparticles included has to be very high for a sufficient osteoinductive effect to take place 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 once formed. This material is totally inorganic, has no organic phase, so its mechanical resilience and resistance to fracture are limited, even more taking into account the porosities achieved. In addition its ability to be biodegraded is also limited.

It follows that none of the three-dimensional macroporous supports described in the prior art meets all the necessary and desirable requirements for its effective and satisfactory application in tissue engineering. Therefore, there is still a need in the state of the art to provide alternative three-dimensional macroporous hybrid supports, which overcome at least part of the disadvantages of the prior art supports.

In this sense the inventors of the present invention have surprisingly discovered that it is possible obtain a new three-dimensional support of polycaprolactone (PCL) and an inorganic phase of biomimetic hydroxyapatite (HAp), by means of a new preparation procedure based on the combination of a method of separation of liquid phases with the extraction of solidified solvent at low temperature with a specific solvent The procedure is reproducible, and allows controlling the pore size of the new support comprised between a few microns to several hundred microns.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1: a graph showing the temperature variation

( ⁰ C) of the cloud point for different homogeneous solutions, depending on the concentration of PLC (between 3% -20%), and the percentage of water (between 10% -15%) in the mixture of water and dioxane solvents .

Fig. 2 a, b, c: SEM micrographs of scaffolds obtained by cold sublimation of ternary dioxane / water-PCL mixtures (of composition 88 / 12-10 with 1% of tween 80) varying the heat treatment time below cloud point

Fig. 3 a, b: SEM micrographs of a scaffold coated with a dense layer of hydroxyapatite after nucleation treatment and a week of immersion in SBF (x5000) Fig. 4 a, b: Microscope image of the preceding sample, and the corresponding X-ray spectrum taken at the point indicated in the image

Fig. 5: SEM micrograph of a scaffold with an average pore size of less than 10 microns Fig. 6: SEM micrograph of a scaffold with an average pore size of more than 50 microns.

OBJECT OF THE INVENTION

In one aspect the present invention relates to a new three-dimensional hybrid support consisting of an organic polymeric polycaprolactone matrix and an inorganic phase consisting of a biomimetic HAp coating. The new hybrid support has a particular morphology with a network of pores perfectly interconnected with each other in size between 5 and 300 microns.

In another aspect the invention relates to a new method of obtaining the hybrid support.

In a further aspect the invention relates to a support obtainable according to the process of the invention.

In another aspect the invention relates to the use of hybrid support in applications such as tissue generation or regeneration, for example, connective tissue, such as bone or cartilage.

In a further aspect, the invention relates to a process for generating or regenerating tissue comprising obtaining a hybrid support according to the invention, and culturing cells of a selected tissue on said support. Optionally, the procedure may also include seeding with support cells.

DESCRIPTION OF THE INVENTION

In one aspect the present invention relates to a three-dimensional macroporous hybrid support, also referred to as "scaffold" or support of the invention, consisting of a porous polycaprolactone matrix (PCL) and a hydroxyapatite coating. biomimetic that covers the internal surfaces of the pores of the matrix. The support has a structure of perfectly interconnected pores, and sizes ranging from 5 to 300 microns. In this regard, Figure 5 shows an SEM photomicrograph of a support according to the invention having pores of less than 10 microns; and Figure 6 shows the SEM photomicrograph of a support with pores larger than 50 microns that can be invaded by cells of very diverse biological tissues.

The adhesion of the biomimetic HAp coating to the polymer matrix is very high and gives the scaffold good mechanical properties. In the context of the present invention ^λλ biomimetic hydroxyapatite "refers to hydroxyapatite that is naturally deposited ^Λλ in vivo" or that is deposited by immersing the support in an equivalent solution that reproduces the conditions "in vivo" of the same composition saline than human plasma (Simulated Body Fluid or SBF) for as long as necessary.

The characteristics of the support of the invention vary depending on its chemical composition and the method of production. In this sense, the support of the invention can have a porosity between 70 and 90%; In addition, the support can have different properties, and can be from spongy, very deformable support, whose plastic deformation allows them to take the form of a pre-existing hole, to elastic supports with modules between 0.05 and 2 MPa.

The support of the invention also has other advantages, which make it especially interesting in tissue applications, such as bone regeneration.

The matrix is PCL, a biodegradable, biocompatible and bioreabsorbable polymer, which is also approved for clinical use. The term "biocompatible" refers in the present invention to being non-toxic and allowing cells to colonize it. The term "bioabsorbable" 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 support of the invention also has good osseointegration and osteoinduction properties; induces the proliferation and differentiation of osteoblasts, induces the formation and calcification of new bone, and restores physiological function at the molecular and cellular levels. In this sense, the support can be implanted "in vivo". Optionally, the support can be seeded with "in vitro" cells prior to implantation.

The structure of the hybrid support is seen in Figures 3 a and b in which it is clearly shown that the PCL matrix, and the biomimetic hydroxyapatite phase intermingle in a highly interpenetrated manner and constitute continuous phases.

In another aspect the invention provides a new method for obtaining the support of the invention comprising the following steps:

(i) preparing a homogeneous solution of PCL in a mixture of two solvents Dl and D2, optionally in the presence of a surfactant; (ii) fill a mold with the homogeneous solution with the shape and dimensions of the support piece to be obtained;

(iii) cooling the solution to a temperature below the temperature at which the separation of two liquid phases occurs;

(iv) maintain the temperature reached in step (iii) until the desired phase morphology is reached;

(v) solidify both phases by lowering the temperature;

(vi) extract solvents Dl and D2 with a third solvent D3 in which the PCL is insoluble, at a temperature below the melting temperature of Dl and D2, and at which D3 is in a liquid state; or alternatively by sublimation in cold (freeze drying) (vii) deposition of biomimetic HAp.

Step (i) is generally carried out at a temperature equal to or greater than room temperature, at which the PCL is soluble in the mixture of Dl and D2 in the proportion D1 / D2 used, obtaining a homogeneous solution of PCL in a mixture of the two solvents Dl and D2. If at room temperature the selected mixture is not homogeneous, it is heated; the person skilled in the art can easily recognize in each case the temperature necessary to obtain a homogeneous solution that will depend on the concentration of PCL, not solvent, and possibly the surfactant. Ambient temperature means between 20 ° C and 25 ° C. The surfactant can be a surfactant conventional, commercial or an amphiphilic diblock. Dl is a solvent of the PCL and D2 is a non-solvent of the PCL. By way of illustration, PCL solvents include, among others, tetrahydrofuran, dimethylsulfoxide (DMSO), methylene chloride, ethyl acetate, chloroform, n-heptane, n-hexane, n-pentane, dioxane, benzene, xylene, naphthalene, dimethylformamide, acetic acid, acetone, and mixtures of the foregoing. Examples of non-solvent are, among others, water, ethanol and mixtures thereof.

In the present description the proportions of Dl, D2 and PCL in the mixture are indicated as follows: X _D I / XD2- Xpoi, with X _D i + X _D 2 = 100, with X _D i and X _D 2 being the proportions by weight of each solvent in the solvent mixture, and X _by the proportion by weight of polymer in the solution. In step (ii) once the mold has been filled with the homogeneous solution, it is homogenized again at the previous temperature to alleviate a possible temperature difference of the mold. The mold has to withstand solvents and sudden temperature changes during the procedure, so it has to be chemically and thermally stable. Generally a resin, polytetrafluoroethylene (PTFE) Teflon or metal mold is used. In general, the mold is closed, and the homogeneous mixture in the closed mold is subjected to the next step.

In step (iii) the homogeneous solution is cooled, and two phases are separated; one of the phases is formed mainly by the dissolution of the PCL in the solvent Dl and the other phase mostly by a mixture of the solvents Dl and D2. At the temperature at which the solution becomes cloudy it is called a "cloud point." The dissolution becomes cloudy as a result of the light scattering in the microscopic nuclei of one phase in the other: it is an indicator of the moment from which the phase phase of the phase diagram is passed to a heterogeneous zone (presence of several phases). The dependence of the "cloud point", which allows setting the rest of the heat treatment conditions, with the composition of the solution, depends on the concentration of PCL and the solvents Dl and D2 used, and can be determined in each case (see Figure one) . The temperature reached in the stage (üi) is lower than that of the cloud point, typically 5 ° C below it.

In stage (iv) the temperature reached in stage (üi) is maintained during. a time generally between 3 and 30 minutes, until reaching the desired phase morphology, that is, until spherical inclusions of poor phase in PCL reach the desired pore size for the scaffold.

The process of liquid phase separation is kinetically controlled, which means that the distribution, size and shape of the two coexisting phases varies depending on the hardening depth below the temperature ^"point" cloud "(iii ) and / or the isothermal treatment time of the mixture at the temperature below the cloud point, in step (iv).

When the system formed by the two phases, still in the liquid phase, reaches the desired morphology, the temperature (stage ((v)) is sharply decreased by solidifying both phases by crystallization of the solvents and partial crystallization of the polymer. Such rapid cooling is desirable. like the one produced by immersion in liquid nitrogen (tempering), to limit the further evolution of the structure.

The solvents Dl and D2 are then extracted with a third solvent D3 in which the PCL is insoluble, at a temperature below the melting temperature of Dl and D2, and at which D3 is in a liquid state. Solvents Dl and D2 are dissolved in D3. D3 can be a low melting alcohol, with the melting point of D3 being much lower than that of D2. In a particular embodiment, the ethanol frozen system is introduced at -20 ° C until complete dissolution of Dl and D2 is achieved, leaving only the PCL with the porous architecture derived from the structure formed during the separation of liquid phases, and proceeds to Dry it under vacuum and at room temperature until complete removal of solvent D3.

Alternatively the extraction can be carried out by cold sublimation (freeze drying)

The final stage of deposition of biomimetic hydroxyapatite on the surface of the support is performed according to the conventional technique of biomimetic hydroxyapatite coating, which consists of depositing the minerals that make up the ceramic bone material from a simulated body fluid (SBF) by immersion of the polymer matrix in said ion-rich fluid with an ion composition close to human plasma as occurs in the body (table 1). Biomimetic hydroxyapatite deposition comprises the following stages:

1) treatment of the support by exposure to a gas plasma or by immersion in a hydroxide solution sodium;

2) immersion the support obtained in 1 alternatively in solutions containing respectively Ca ²⁺ or PO4 ^{3 ~} ;

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

Step 1) introduces carboxyl groups on the surface of the pores of the PCL, suitable for nuclear calcium phosphate. Then in step 2) calcium phosphate cores are generated on the surface of the micropores and macropores of the support, by alternating immersion of the support obtained in 1) in solutions containing respectively Ca ²⁺ and PO ₄ ^{3 ""} , for example calcium chloride and potassium phosphate. In step 3) the support obtained in 2) is immersed in a simulated body fluid for a period of time between a few days and two weeks. The longer the time, the greater the thickness of the biomimetic hydroxyapatite coating layer created, depending on the required application. Alternatively, the support obtained in 2) can be immersed in another modified solution. The composition of the simulated body fluid (SBF) can be modified to accelerate the deposition process or to include ions that are not present in the normal environment but may have osteoinductive properties (for example fluorine).

In a preferred embodiment the following ionic composition of the simulated body fluid (SBF) buffered with hydrochloric acid and tris (hydroxymethyl) aminomethane hydrochloride of Table 1 is used:

Table 1

The support is submerged for a period typically between 24 hours and 2 weeks. Depending on the time, a coating of hydroxyapatite of variable thickness is generated (the greater the longer the deposition time) that completely covers the surface of rαacro and micropores. A hybrid support is thus obtained with an interpenetrated structure as described and illustrated in the invention. As the SEM photomicrograph images (Figures 3 and 4) show, the deposited biomimetic hydroxyapatite covers all internal surfaces of the pores, constituting a continuous phase, and originating the interpenetrated hybrid structure of the support of the invention.

The support is then washed with distilled water and dried, for example in a vacuum desiccator. It can be sterilized according to any conventional method, stored or conditioned for use.

To proceed to a liquid-liquid phase separation, it is necessary to know the phase diagram of the solvent / non-solvent / PCL ternary system chosen. Also in the process of the invention results decisive to control the relationship between the composition of the solution in terms of the weight fractions of PCL and of the solvents Dl and D2 ^' (Xm, XD2, Xpoi) ^{and the} heat treatment and phase morphology to obtain precisely and reproducible a support according to the invention. This is illustrated in examples 1 and 2.

The characteristics of the support of the invention can be varied in a simple manner by one skilled in the art within the ranges described in the present invention to adapt to the needs required by the specific application. Figures 5 and β show how very variable porous structure architectures can be obtained by this procedure, from structures with small pores, some microns, which are perfectly interconnected (Figure 5), to large pores that can be invaded by cells of very diverse biological tissues (Figure 6).

As already mentioned above, the hybrid supports of the invention are suitable for application in tissue engineering.

Therefore in another aspect the invention provides the use of the support of the invention for its use in tissue applications, in the generation or regeneration of tissue. For non-complex "scaffold" forms, various cutting tools can be used after obtaining them to obtain the desired geometry for their specific use.

The generation or regeneration of tissue includes bone repair or replacement, intervertebral fusion strips, prosthetic implant in bone tissue "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 thus relates to a method for generating or regenerating tissue comprising: providing a hybrid support according to the present invention, and culturing in vitro in said support cells of a tissue, and optionally implanting the support obtained in the previous stage in a human or animal.

Optionally, the procedure can also comprise seeding with support cells prior to their culture and implantation.

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

Likewise, the present invention relates to a method of treatment for the generation or regeneration of a tissue in a subject comprising implanting a hybrid support in said subject in need thereof, where the 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 illustrative examples 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 1. Preparation of a scaffoid

Example 1. Determination of the phase diagram of dioxane / water / polymer solutions.

Several dioxane / water-PCL solutions were prepared, varying both the concentration of PCL between 3% and 20% and that of water in the mixture of water and dioxane solvents between 10% and 15%. The mixtures are heated until they are in the single-phase zone, where the solution has a transparent and homogeneous appearance. This zone, where the dioxane-water-PCL ternary mixture is homogeneous, is located above the curves of Figure 1. The temperature was then gradually lowered, leaving at a given temperature 10 minutes, observing, and lowering another degree, etc. The points of the curves indicate the temperature at which the phase separation begins for a given system. Figure 1 shows how for different concentrations of PCL between 3% and 20%, and for different percentages of water the temperature of the "cloud point" varies. In this sense, the cloud point increases strongly and linearly with the proportion of non-solvent (water) in the mixture, it is more difficult to dissolve the polymer because there is less affinity between the liquid and the polymer. The same is true, although to a lesser extent, for the concentration of polymer: the more polymer in the solution, the more difficult it is to dissolve it, so the curve with the highest concentration (20% PCL) is superior to all others. For all series, the observed trend is the same, and for a narrow range of concentrations, the slope of the curve does not change significantly (Cf. concentrations of 3, 5, 8%). The dotted box represents the temperature range where you can work comfortably. It is not possible to work at high temperatures, since the liquid-vapor equilibrium of the solvent shifts from the steam side, and consequently the concentration of the mixture is altered. Nor is it desirable to work at lower temperatures than the environment, as it requires more equipment and more refined temperature control; In addition, at low temperatures, both the crystallization of dioxane (Tf _US ion: 11 ⁰ C) and the polymer gel (crystallization in solution) can occur, which ultimately produces solidification, preventing the separation of liquid-liquid phases for the benefit of a liquid-solid phase separation.

The attachment of any surfactant, thickener, or other substance, modifies the phase equilibrium, so the diagram has to be recalculated.

Example 1.2: Influence of nucleation time on the scaffold structure

Likewise, the microstructure of the support obtained has been determined as a function of the isothermal treatment time, (3, 10 and 15 minutes) in particular at 5 ° C below the cloud point (Figure 2 a, b, c) for systems 88 / 12-10 (dioxane / water-PCL) and 1% tweenδO. The dioxane-water-PCL 88 / 12-10 system is a solution containing 10% by weight of PCL in a mixture of dioxane and water in which the percentage of dioxane is 88% by weight.

A dioxane / water mixture in proportion 88/12 was prepared. 10% by weight was added to the liquid PCL respect, and 1 wt% Tween 80. The cloud point temperature of this mixture was determined by the procedure described in Example 1.1, and a value of 37 ⁰ C was obtained The mixture is homogenized for fifteen minutes in the monophase region at 40 ⁰ C, then cooled in a bath at 32 ° C, in the two - phase zone, T _cloud -5 ^O C. was left in this zone for the separation of liquid-liquid phase occurred during 3, 10 and 15 rain and the influence of nucleation time on the pore size obtained was observed. It was then cooled sharply by immersion in liquid nitrogen, which freezes the structure as it is. Then the solvent (water dioxane mixture) was extracted at low temperature by another solvent that is not solvent of the polymer, in this case ethanol. The extraction ethanol was changed three times, and then the sample was dried.

As can be seen in Figures 2 a, b and c, the longer the nucleation time, the greater the size of the pores obtained. In any of the times used, however, the interconnection between the pores formed is observed.

Example 1.3: Nucleation of an inorganic phase similar to bone apatite to obtain the hybrid structure

A scaffold was synthesized with a mixture of 88/12 dioxane and water, and 8% by weight of PCL with respect to the solvent weight, with a nucleation time of 3 minutes at

The scaffold was treated with a 1M solution of sodium hydroxide for 24 hours at room temperature. It was then treated with solutions of calcium chloride (0.2 M) and potassium phosphate (0.2M) to generate nuclei of calcium phosphate on the surface.

After the treatment, the scaffold was immersed in simulated body fluid, (SBF, Simulated Body Fluid), an aqueous solution whose molar composition is described in Table 2. After one week the scaffold was washed with water distilled, and then with ethanol and dried. The micrographs presented (Fig 3a and 3b) show the abundant nucleation of a mineral layer on top of the synthetic support. The EDX analysis confirms that this mineral layer has a composition similar to hydroxyapatite, bone mineral, due to its richness in calcium phosphate and its impurities (magnesium, potassium) typical of biological hydroxyapatite.

Table 2: Ionic composition of simulated body fluid and blood plasma, (mM / 1) A: Simulated body fluid; B: blood plasma.

Claims

1. A macroporous and three-dimensional support consisting of a porous polycaprolactone matrix and a biomimetic hydroxyapatite coating that covers the internal surfaces of the pores, interconnected with each other, and of sizes between 5 and 300 microns, in which the matrix Polycaprolactone porous and hydroxyapatite coating intermingle and constitute continuous phases.

2. Support according to claim 1, which has a volume porosity between 70 and 90%.

fifteen

3. Support according to any of claims 1 to 2, which has an elastic module between 0.5 MPa and 2MPa.

A method for obtaining a support according to any one of claims 1 to 3, comprising the steps of:

(i) preparing a homogeneous solution of PCL in a mixture of two solvents, a PCL solvent, and a non-2.5 PCL solvent, optionally in the presence of a surfactant;

(ii) filling a mold with the homogeneous solution obtained in the previous stage;

(iii) cooling the homogeneous solution to a temperature below the temperature at which the separation of two liquid phases occurs; (iv) maintain the temperature reached in the previous stage until the desired phase morphology is reached; (v) solidify both phases by lowering the temperature; (vi) extract the PCL solvent and the PCL non-solvent, with a third solvent in which PCL is insoluble, at a temperature below the melting temperature of the PCL solvent and the PCL non-solvent, and that the third solvent is in a liquid state;

(vii) deposition of biomimetic HAp.

5. The method according to claim 4, wherein step (i) is carried out at a temperature equal to or greater than the ambient temperature.

6. The process according to claim 4 or 5, wherein the PCL solvent is selected from the group consisting of tetrahydrofuran, dimethyl sulfoxide, methylene chloride, ethyl acetate, chloroform, n-heptane, n-hexane, n-pentane, dioxane , benzene, xylene, acetone, naphthalene, dimethylformamide, dioxane, acetic acid, acetone and mixtures thereof.

7. A process according to claim 4 or 5, wherein the non-solvent is selected from the group consisting of water, ethanol and mixtures thereof.

8. Process according to claims β and 7, wherein the solvent is dioxane and the non-solvent is water.

9. The method according to claim 8, wherein the concentration of water in the mixture of dioxane and water is between 10% and 15%, and in which the concentration of PCL is between 3% and 20%.

10. The method according to claim 9, wherein the homogeneous solution is prepared from a mixture of dioxane / water in 88/12 proportion, 10% by weight of PCL with respect to the solvent mixture and 1% tween 80.

11. A method according to any one of claims 4 to 10, wherein the homogeneous solution is cooled 5 ° C below the cloud point temperature, and is maintained at said temperature for a time between 3 and 30 minutes.

12. The method according to any one of claims 4 to 11, wherein step (v) is carried out by immersion of the system formed by the two phases in liquid nitrogen.

13. Process according to any one of claims 4 to 12, wherein step (vi) is carried out in ethanol at -20 ⁰ C.

14. The method according to claim 4, wherein the extraction stage (vi) is carried out, alternatively, by cold sublimation.

15. The method according to any one of claims 4 to 14, wherein the step (vii) comprises the following steps:

1) treatment of the support by exposure to a gas plasma or by immersion in a solution of sodium hydroxide. 2) immersion the support obtained in 1) alternatively in solutions containing respectively each Ca ²⁺ ions and PO4 ^{3 "} ions.

3) immersion of the support obtained in simulated body fluid.

16. Support obtainable according to any of claims 4 to 15.

17. Support according to any of claims 1 to 3 for use in tissue generation or regeneration.

18. Use of the support according to any of claims 1 to 3, for tissue generation or regeneration.

19. Use according to claim 18, wherein the tissue is connective.

20. Method for generating or regenerating tissue comprising: obtaining a hybrid support according to any one of claims 1 to 3, and culturing in vitro in said support cells of a tissue, and optionally planting with cells of the support prior to its cultivation.