ES2330823B2 - CO-CONTINUOUS HYBRID STRUCTURE FOR THE REGENERATION OF OSE DEFECTS. - Google Patents

Abstract

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

The present invention describes a process to obtain a scaffold consisting of a porous matrix PCL and a hydroxyapatite coating comprising: (i) prepare a homogeneous solution of PCL in a mixture of two solvents, a PCL solvent, and a non-solvent of PCL, (ii) filling a mold with said homogeneous solution; (iii) cool the homogeneous solution to a temperature below the temperature at which the separation of two liquid phases occurs; (iv) isothermal treatment; (v) solidify both phases lowering the temperature; (vi) extract the solvent from PCL and the non-solvent PCL, with a third solvent; Y (vii) deposition of biomimetic HAp. The support is useful in tissue engineering applications.

Description

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

Field of the Invention

The present invention fits within the field of tissue engineering, and more particularly refers to a three-dimensional macroporous support, comprising a matrix polycaprolactone porous and a hydroxyapatite coating biomimetics The invention also relates to the process of obtaining such support, as well as its use in Applications such as bone tissue regeneration.

Background of the invention

Due to the suffering of cancers, accidents or due to bone degeneration a massive loss of bone tissue, and give rise to the need for a bone graft. A Ideal bone graft to repair bone defects should allow bone cells grow in the affected area, to restore the function and physical integrity of the bone. Today, autografts they are the preferred option for bone repair because they are biocompatible and there is no risk of disease transmission. Without However, the limitations of autografts are low amount of tissue obtainable, the double operation necessary to carry out the graft, and morbidity at the site of extraction, usually associated with discomfort and suffering for the patient. Allografts, which consist of tissue grafting extracted from the bone of a corpse, they are also solutions but it increasingly rule out the risk of disease transmission and immunological rejection.

To solve the problems associated with bone grafts, many researchers have tried to develop artificial substances that can serve as bone graft. At state of the art defines the qualities that such must possess material to be implanted successfully. First, the material must be biodegradable so that as the new bone goes forming can grow at the graft site, since it goes disappearing over time. Both the material and its products of degradation must be biocompatible, so as not to present a risk Some for the patient's health. Second, the material must maintain mechanical characteristics similar to those of bone, so that the loads transmitted in the implant favor the bone tissue 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. materials osteoconductive include calcium phosphate-based ceramics, such as hydroxyapatite (natural inorganic component of bone), the Calcium triphosphate or biovírios (Bioglass ®).

Ceramics and bioglasses are compatible by the similarity of its chemical structure with that of the native bone, without however they are difficult to process, especially in porous form, and not they have the flexibility that gives the bone its organic part, the collagen, being very rigid and brittle. In addition, the Sintered hydroxyapatites are highly crystalline and do not degrade easily, which in the long term can prevent a complete bone regeneration.

Biodegradable polymers that wake up a lot interest in the field of tissue engineering are for example the polycaprolactone (PCL), polylactic acid (PLA), acid polyglycolic (PGA) acid (lactic-co-glycolic), etc. They are easy to shape and transform, and degrade at a speed which can be adapted to the bone regeneration rate. Although you can synthesize scaffolds from them easily and get them to have good mechanical properties, pure polymers  They are not ideal for this use since they are not very osteoconductive. The neoformed bone will not adhere or grow well in the pure materials

Due to this fact, of course, 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. Various studies have demonstrated the feasibility of this approach (Biomaterials, in press; Fabrication of three-dimensional polycaprolactone / hydroxyapatite tissue scaffolds and osteoblast-scaffold 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 numerous works procedures are raised so that biomimetic hydroxyapatite can be nuclear in the surface of the polymeric material in order to increase adhesion Cellular and bioactivity. The presence of hydroxyapatite biomimetic grafting may favor bone remodeling by redisolution of the deposited apatite. Additionally and due to a greater excess of ions in the middle, the presence of the hydroxyapatite may serve to regulate the pH of the surrounding environment of the graft since some degradation products of the polymers are acidic (for example in the case of acid polylactic). Techniques have been developed to coat biomimetic hydroxyapatite different polymeric materials always that these have hydroxyl functional groups such as the 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 you to intuit that the properties The biological aspects of such apatite are significantly better than those of calcium phosphates or traditional synthetic apatites. The Causes that explain these best results are varied. Between others highlights that the average size of the apatite crystal formed by This procedure is small, which favors the redisolution of The crystals in the body. This is a phenomenon that does not it is observed with sintered synthetic apatites at high temperatures, which are highly stable and do not decompose. Other cause is that the chemical composition of this type of apatite is much more similar to that of natural bone.

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

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 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 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, which comprises bioactive microparticles that could induce tissue Human bone to regenerate. The support uses the combination of microparticles of silica, calcium, phosphorus as substances bioactives that could actively induce proliferation and differentiation of human osteoblasts, promoting formation and calcification of new bone. However the volume of microparticles included has to be very high for  that a sufficient osteoinductive effect take place in 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 material is totally inorganic, it has no 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

It follows that none of the three-dimensional macroporous supports described in the state of the technique, meets all necessary and desirable requirements for its effective and satisfactory application in tissue engineering. Therefore, there is still a need in the state of technique of providing macroporous hybrid supports three-dimensional alternatives, which exceed 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 to obtain a new three-dimensional polycaprolactone (PCL) support and a 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 solvent solidified at low temperature with a solvent specific. The procedure is reproducible, and allows control the pore size of the new support comprised between some microns to several hundred microns.

Brief description of the figures

Fig. 1: a graph showing the variation of temperature (ºC) of the cloud point for different solutions homogeneous, depending on the concentration of PLC (between 3% -20%), and of the percentage of water (between 10% -15%) in the solvent mixture Water and dioxane.

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

Fig. 3 a, b: SEM photomicrographs of a scaffold coated with a dense layer of hydroxyapatite after treatment of  nucleation and a week of immersion in SBF (x5000)

Fig. 4 a, b: Microscope image of the sample preceding, and the corresponding X-ray spectrum taken in the point indicated in the image

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

Fig. 6: SEM micrograph of a scaffold with a average pore size greater than 50 microns.

Object of the invention

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

In another aspect the invention relates to a new procedure for obtaining hybrid support.

In a further aspect of the invention, refers to a support obtainable according to the procedure of the invention.

In another aspect the invention relates to use of hybrid support in applications such as 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 hybrid support according to the invention, and cultivating on said support cells of a selected tissue. Optionally the procedure may also include seeding with cells of the support.

Description of the invention

In one aspect the present invention relates to a three-dimensional macroporous hybrid support, also called hereinafter "scaffold" or support of the invention, which consists in a porous polycaprolactone matrix (PCL) and a coating of biomimetic hydroxyapatite lining the internal surfaces of the pores of the matrix. The support has a structure of pores perfectly interconnected with each other, and of sizes between 5 and 300 microns. In this regard, Figure 5 shows an SEM photomicrograph of a support according to the invention that presents 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 tissues  Biological

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 "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 composition saline than human plasma (Simulated Body Fluid or SBF) for as long as necessary.

The characteristics of the support of the invention,  they vary depending on their chemical composition and the procedure of obtaining. In this sense the support of the invention can present a porosity between 70 and 90%; also the support can present different properties, and be from support fluffy, very deformable, whose plastic deformation allows them take the form of a pre-existing hole, until elastic supports  with modules between 0.05 and 2 MPa.

The support of the invention also presents other  advantages, which make it especially interesting in applications tissue, 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 observed in Figures 3 a and b in which it is clearly shown that the matrix PCL, and the biomimetic hydroxyapatite phase intermingle with highly interpenetrated manner and constitute continuous phases.

In another aspect the invention provides a new method for obtaining the support of the invention that It comprises the following stages:

(i) prepare a homogeneous solution of PCL in a mixture of two solvents D1 and D2, optionally in the presence of a surfactant;

(ii) fill with the homogeneous solution a mold with the shape and dimensions of the support piece that want to get;

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

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

(v) solidify both phases by decreasing the temperature;

(vi) extract solvents D1 and D2 with a third  solvent D3 in which the PCL is insoluble, at a temperature below the melting temperature of D1 and D2, and at which D3 is it 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 PCL is soluble in the mixture of D1 and D2 in the D1 / D2 ratio used to obtain a homogeneous solution of PCL in a mixture of the two solvents D1 and D2. If at room temperature the Selected mixture is not homogeneous heated; the expert in matter can easily recognize in each case the temperature necessary to obtain a homogeneous solution that will depend on the  PCL concentration, non-solvent, and eventually the surfactant. Ambient temperature means between 20ºC and 25ºC. He surfactant can be a conventional, commercial surfactant or a amphibyl diblock. D1 is a solvent of the PCL and D2 is a non-solvent PCL. By way of illustration among PCL solvents can be mentioned 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 above. Examples of Non-solvent are among others water, ethanol and their mixtures

In the present description the proportions of D1, D2 and PCL in the mixture are indicated as follows: X_ {D1} / X_ {D2} - X_ {pol}, with X_ {D1} + X_ {D2} = 100, being X D1 and X D2 the proportions by weight of each solvent in the solvent mixture, and X pol the proportion by weight of polymer in the dissolution.

In step (ii) once the mold has been filled with the homogeneous solution, it is homogenized again at previous temperature to alleviate a possible difference of mold temperature. The mold has to withstand the solvents and sudden temperature changes during the procedure, by which has to be chemically and thermally stable. Usually a resin mold, of polytetrafluoroethylene (PTFE) is used Teflon or metal. In general the mold is closed, and the mixture Homogeneous in the closed mold is subjected to the next stage.

In step (iii) the homogeneous solution is it cools, and two phases separate; one of the phases is formed mostly by dissolving the PCL in solvent D1 and the another phase mostly by a mixture of solvents D1 and D2 At the temperature at which the solution becomes cloudy it will be called "cloud point." The solution becomes cloudy as consequence of light scattering in microscopic nuclei from one phase to the other: it is an indicator of the moment where it passes from the single phase zone of the phase diagram to a heterogeneous zone (presence of several phases). The dependence of the "point of cloud ", which allows to fix the rest of the treatment conditions thermal, with the composition of the solution, depends on the concentration of PCL and the solvents D1 and D2 used, and can determined in each case (see Figure 1). Temperature reached in step (iii) it is lower than that of the cloud point, typically 5 ° C below this.

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

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

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

Solvents D1 and D2 are then extracted with a third solvent D3 in which the PCL is insoluble, at a temperature below the melting temperature of D1 and D2, and at the that D3 is in a liquid state. Solvents D1 and D2 are dissolved in D3. D3 can be a low melting alcohol, being mandatory to have the melting point of D3 well below of D2. In a particular embodiment the system is introduced frozen in ethanol at -20 ° C until complete dissolution of D1 and D2, leaving only the PCL with the porous architecture derived from the structure formed during phase separation liquid, and it is dried under vacuum and at room temperature until complete removal of solvent D3.

Alternatively the extraction can lead to out by cold sublimation (freeze drying).

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

one)

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

2)

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

3)

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

Stage 1) introduces on the surface of the PCL pores carboxyl groups, suitable for nuclear phosphate calcic. Then in stage 2) nuclei of calcium phosphate on the surface of 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 fluid simulated body for a period of time between Days and two weeks. The longer the time, the greater the layer thickness biomimetic hydroxyapatite coating created, according to the required application. Alternatively the support obtained in 2) can be immersed in another modified solution. The Simulated body fluid composition (SBF) can be modified to accelerate the deposition process or to include ions that do not they are present in the normal environment but may have properties osteoinductive (for example fluoride).

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

TABLE 1

one

The support is submerged for a while typically between 24 hours and 2 weeks. Depending on  time a thick hydroxyapatite coating is generated variable (the greater the longer the deposition time) that covers Totally macro and micropore surface. You get a hybrid support with an interpenetrated structure as described and illustrates in the invention. As the images of SEM photomicrograph (Figures 3 and 4) biomimetic hydroxyapatite deposited covers all internal surfaces of the pores, constituting a continuous phase, and originating the hybrid structure interpenetrated with the support of the invention.

Then the support is washed with water distilled and dried, for example in a vacuum desiccator. May sterilize according to any conventional method, be stored or conditioned for use.

To proceed to a phase separation liquid-liquid, it is necessary to know the diagram of Ternary solvent / non-solvent / PCL system phase Let him choose. Also in the process of the invention results determinant control the relationship between the composition of the solution in terms of weight fractions of PCL and of the solvents D1 and D2 (X_ {D1}, X_ {D2}, X_ {pol}), the treatment thermal and phase morphology to obtain accurately and A support according to the invention is reproducible. This is illustrated in the Examples 1 and 2.

The characteristics of the support of the invention can be varied easily by a person 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 6 show how they can be obtained by this procedure very variable porous structure architectures, from structures with small pores, some microns, which are perfectly interconnected (Figure 5), up to large pores size that can be invaded by cells of very diverse tissues  biological (Figure 6).

As mentioned earlier, the brackets hybrids 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 use tissue applications, in the generation or regeneration of tissue. For non-complex forms of "scaffold" can be used after obtaining, various cutting tools for Obtain the desired geometry for your specific use.

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 hybrid support according to the present invention, and

culturing tissue cells 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.

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 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.

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Examples 1. Preparation of a scaffold Example 1 Determination of the solution phase diagram dioxane / water / polymer

Several solutions were prepared dioxane / water-PCL, varying both the concentration of PCL between 3% and 20% as the water in the solvent mixture water and dioxane between 10% and 15%. The mixtures are heated until be in the single phase zone, where the solution has an aspect transparent and homogeneous. This area, where the ternary mix dioxane-water-PCL is homogeneous, it It is located above the curves of Figure 1. The following is the temperature dropped gradually, leaving at a given temperature 10 minutes, observing, and lowering another grade, etc. The points of the curves indicate the temperature at which the separation of phases for a given system. Figure 1 shows how for different PCL concentrations between 3% and 20%, and for different percentages of water varies the temperature of the "point of cloud. "In this sense, the cloud point increases sharply and linearly with the proportion of non-solvent (water) in the mixture, it costs more to dissolve the polymer because there is less affinity between the liquid and the polymer. The same happens, although in lower grade, for polymer concentration: the more polymer there is in the solution, it is more difficult to dissolve it, so the curve with the highest concentration (20% PCL) it 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 changes significantly (Cf. concentrations of 3, 5, 8%).

The dotted box represents the interval of temperatures where you can work comfortably. It can not work at high temperatures, because the balance liquid-vapor solvent moves from the side of the steam, and consequently the concentration of the mixture is altered. It is also not desirable to work at temperatures lower than the environment, because it requires more equipment and a control of more refined temperature; also at low temperatures you can produce both dioxane crystallization (T fusion: 11 ° C) as the polymer gel (crystallization in solution) that in definitively produces a solidification, preventing the separation of liquid-liquid phases for the benefit of a liquid-solid phase separation.

The attachment of any surfactant substance, thickener, or other, modifies the phase equilibrium, so You have to recalculate the diagram.

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Example 1.2 Influence of nucleation time on the structure of the scaffold

It has also been determined how the microstructure of the support obtained based on the time of isothermal treatment, (3, 10 and 15 minutes) in particular at 5 ° C for below the cloud point (Figure 2 a, b, c) for systems 88 / 12-10 (dioxane / water-PCL) and 1% of tween80. The system dioxane-water-PCL 88 / 12-10 is a solution that contains 10% in weight of PCL in a mixture of dioxane and water in which the percentage Dioxane is 88% by weight.

A dioxane / water mixture was prepared in ratio 88/12. 10% by weight of PCL was added with respect to the liquid, and 1% by weight of Tween 80. The temperature of the cloud point of such mixture was determined by the procedure described in the Example 1.1, and a value of 37 ° C was obtained. The mixture was homogenized during a quarter of an hour in the single phase zone at 40 ° C, then cooled in a bath at 32 ° C, in the biphasic zone, to T_ {cloud} -5ºC. It was left in this area so that the liquid-liquid phase separation occurs, for 3, 10 and 15 min and the influence of the time of nucleation on the pore size obtained. Then it cooled sharply by immersion in liquid nitrogen, which freeze the structure as it is. Then he proceeded to the solvent extraction (dioxane water mixture) 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 seen in Figures 2 a, b and c The longer the nucleation time, the larger the size of the pores obtained. At any of the times used, However, it observes the interconnection between the pores formed.

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

A scaffold was synthesized with a mixture of 88/12 of dioxane and water, and 8% by weight of PCL with respect to the weight of solvent, with a nucleation time of 3 minutes at T_ {cloud} -5.

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

After the treatment the scaffold was submerged in simulated body fluid, (SBF, Simulated Body Fluid), a aqueous solution whose molar composition is described in the Table 2.

After a week the scaffold was washed with distilled water, and then with ethanol and dried. Micrographs presented (Fig 3a and 3b) show the abundant nucleation of a mineral layer above the synthetic support. He EDX analysis allows to confirm that this mineral layer has a hydroxyapatite-like composition, bone mineral, by its richness in calcium phosphate and its impurities (magnesium, potassium) typical of the biological hydroxyapatite.

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

2

Claims (14)

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1. A process for obtaining a macroporous and three-dimensional support consisting of a porous polycaprolactone matrix and a biomimetic hydroxyapatite coating that covers the internal surfaces of the matrix pores, characterized in that it comprises the steps of:

(i)

prepare a homogeneous solution of polycaprolactone in a mixture of two solvents, a solvent of polycaprolactone and a non-solvent of polycaprolactone and, optionally, in the presence of a surfactant;

(ii)

fill a mold with the homogeneous solution obtained in the previous stage;

(iii)

cool the homogeneous solution to a temperature below the temperature at which the separation of two liquid phases;

(iv)

maintain the temperature reached in the stage anterior until the morphology of the phases is reached desired;

(v)

solidify both phases by decreasing the temperature;

(saw)

extract the polycaprolactone solvent and the non-solvent polycaprolactone with a third solvent in which polycaprolactone is insoluble, at a temperature below the melting temperature of the solvent of polycaprolactone and non-solvent polycaprolactone, and to which the third solvent is found in liquid state;

(vii)

deposition of biomimetic HAp.

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2. Method according to claim 1, in which stage (i) is carried out at an equal temperature or Ambient temperature exceeds. 3. Method according to claim 1 or 2, wherein the polycaprolactone solvent is selected from the group formed by tetrahydrofuran, dimethylsulfoxide, methylene chloride, ethyl acetate, chloroform, n-heptane, n-hexane, n-pentane, dioxane, benzene, xylene, acetone, naphthalene, dimethylformamide, dioxane, acetic acid, acetone and mixtures thereof. 4. Method according to claim 1 or 2, in which the non-solvent is selected from the group formed by water, ethanol and mixtures thereof. 5. Method according to claims 3 and 4, in which the solvent is dioxane and the Non-solvent is water. 6. Method according to claim 5, in that the concentration of water in the mixture of dioxane and water is between 10% and 15% and in which the concentration of Polycaprolactone is between 3% and 20%. 7. Method according to claim 6, in which the homogeneous solution is prepared from a mixture of dioxane / water in proportion 88/12, 10% by weight of polycaprolactone with respect to the mixture of solvents and 1% of tween 80. 8. Procedure according to any one of the claims 1 to 7, 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 9. Procedure according to any one of the claims 1 to 8, wherein step (v) is carried out by immersion of the system formed by the two phases in nitrogen liquid. 10. Procedure according to any one of the claims 1 to 9, wherein step (vi) is carried out in ethanol at -20 ° C. 11. Method according to claim 1, in which stage (vi) of extraction is carried out, alternatively, by cold sublimation. 12. Procedure according to any one of the claims 1 to 11, wherein step (vii) comprises the following stages:

one.

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

2.

immersion the support obtained in (1) alternatively in solutions containing respectively each a Ca2 + ions and PO4-3- ions;

3.

immersion of the support obtained in body fluid simulated.