ES2335851B1 - METHOD FOR LOW TEMPERATURE PREPARATION OF BIOCERAMIC PARTS WITH THREE-DIMENSIONAL POROSITY DESIGNED AND INTERCONNECTED. - Google Patents

Abstract

Method for low temperature preparation of bioceramics pieces with three-dimensional porosity designed e interconnected

The method is based on the gelation of a aqueous suspension of a ceramic and a biocompatible binder in The inside of a mold. The consolidation process occurs at a temperature between 30 and 45 ° C, depending on the formulation employee. The method allows the introduction, during the process of manufacture of biological or pharmacologically active substances. The mold design, consisting of a network of filaments rigid, basically determines the porosity of the piece, although This may be affected by other factors (drying process, composition of the mixture).

Pore size, geometry and percentage Porosity can be modified in the three directions of the space. The pore size that can be achieved with this method (between 300 and 1000 µm) as well as the fact that the pores are interconnected facilitates the vascularization process and, consequently, the integration of the implanted material and, in the Most cases, subsequent tissue regeneration.

The last purpose of these pieces is their application as implants for the regeneration of bone tissue in engineering tissue.

Description

Method for low temperature preparation of bioceramics pieces with three-dimensional porosity designed e interconnected

Technical sector

The present invention fits within the Technical field of implant manufacturing in orthopedic surgery for bone defect filling, for cell growth media in tissue engineering (Tissue Enginneering Scaffolds) and how bioreactors The proposed preparation method allows the incorporation of biological or pharmacologically active substances being able to act as controlled release systems.

State of the art

The application of tissue engineering to the therapeutic repair of bone tissues has become a promising solution. This entails sowing and adhering in vivo human cells on a structure. The cells, once implanted, proliferate, migrate and differentiate into specific tissue while secreting components necessary to create the required tissue. The choice of the structure to be implanted is crucial to allow the cells to behave in a suitable way to produce tissues of a certain shape and size. The materials used for bone tissue regeneration include both synthetic and inorganic natural ceramic materials, such as hidoxyapatite and tricalcium phosphate since these ceramics simulate the natural composition of the bone.

In addition to its composition, the porosity of the structure is a fundamental aspect to allow the penetration of blood, oxygen and nutrients necessary for the cells. Without However, the conventional manufacturing techniques of these structures are not able to control the pore size, the pore geometry, spatial distribution, and, above all, the construction of interconnected internal channels that allow blood, oxygen, nutrients, etc. circulate through all the structure.

There are numerous works that describe methods of obtaining bioceramics pieces with a relatively porosity controlled. Thus, document US2004 / 0002770 describes a method of obtaining ceramics with porosity controlled by compression of a polymer on the porous ceramic material. Although you can achieve a homogeneous distribution of pores in terms of shape and size, the interconnection between the pores is not equally controllable.

There is currently a growing interest in developing new prototyping techniques that allow controlling the architecture of the piece, that is, the shape, size and interconnections of the pores. Among these techniques are Silicon Micromachining, which consists of depositing layers of material on a silicon wafer with a predetermined shape ( Microfabrication Technology for Vascularized Tissue Engineering. Borenstein et al., Biomedical Devices 4: 3 (2002), 167-175 ) or Fused Deposition Modeling (FDM) modeling where three-dimensional objects are constructed directly from 3D CAD data; A temperature controlled head extrudes thermoplastic material in layers, forming a three-dimensional structure by superposition of layers. As a thermoplastic material, polycaprolactone is usually used, which is a bioreabsorbable polymer ( Fused Deposition modeling of novel scaffold architectures for tissue engineering applications . Zein et al. Biomaterials 23 (2002), 1169-1185). One of the most used techniques is the so-called Manufacturing of Free Form Solids (SFF), based on the use of a three-dimensional porogen or template produced from sintered meshes of nylon fibers or another similar polymer, which makes that the matrix resulting from the infiltration and polymerization of the crosslinking material is the inverse or negative structure of the template, achieving a great geometric regularity of interconnected pores and channels. To get this negative, the template must be deleted.

The work published by Liang and Weng ( Artificially controlling of inner structure to porous hydroxyapatite ceramic by using solidified coated fibers , Material Letters 60 (2006) 3569-3572) describes the manufacture of hydroxyapatite ceramic pieces from a three-dimensional fiber structure that It is built to cause a certain distribution of pore sizes and connectivity. A binder and hydroxyapatite suspension is added to this fiber structure. Once the piece is consolidated, the fiber structure is removed by pyrolysis.

US 2004/0062809 describes a process of preparing a biocompatible polymer with a structure Three-dimensional and porosity designed. To do this, use a mold on which the biocompatible material is formed. Once finished such formation, the mold is removed by dissolution (using a solvent) or by enzymatic digestion.

In a previous work published by the inventors of this invention (Cabañas et al. Room Temperature synthesis of agarose / sol-gel glass pieces with tailored interconnected porosity , J. Biomedical Materials Research Part A, 2006) a method of obtaining parts is described of porosity designed from three-dimensional polystyrene molds on which an agarose suspension (binder) and a bioactive ceramic material are poured. Once the ceramic piece is consolidated, the mold is removed by treatment with an aqueous solution of soda.

The methods described above based on mold manufacturing techniques with controlled porosity get obtain parts with a distribution of size, shape and interconnection of the appropriate pores. However, the elimination stage of mold involves a thermal or chemical treatment that can damage the structure of the ceramic piece. On the other hand, these treatments make the inclusion of a biological substance or pharmacologically active within the ceramic piece that will act as an implant, it must be done once the piece is obtained which can have the disadvantage of not being distributed homogeneously to throughout the entire structure and also makes it difficult to control the amount of substance to include.

EP 1 449 818 describes a process for obtaining a porous structure of sintered calcium phosphate consisting of mixing powders of calcium phosphate precursors and pressurizing them between 5 and 500 MPa on a metal, wood or bamboo rod that is then removed. A group of rods are arranged on the same plane in parallel in the same direction; over this group another overlaps in the same direction or in a different direction. Finally, more groups of rods can be added perpendicular to the first groups. Once the powders are compacted, the rods are removed giving rise to a porous piece. In order for the piece to have an appropriate hardness it must be synthesized at an elevated temperature, between 500 and 1300 ° C. US 2005/0025807 describes a process to obtain a calcium phosphate setting cement that also uses the same rod system as a mold to obtain a porous material. After setting, the material must be heated between 300 and 500 ° C to obtain the appropriate hardness, unless a biocompatible polymer (especially collagen) is used as the binding agent. It can also be included in drug in the mixture to be set to form the cement. In 2005, Ito ( Three-dimensionally perforated phosphate ciacium phosphate ceramics. J. Wuhan University of Technology - Mat. Sci. Ed. , 20. Suppl.) Described the same rod procedure to produce porous calcium phosphate ceramic by compression of a solution of precursor powders at a pressure of 36 MPa and subsequently synthesized at temperatures close to 1000 ° C; It also uses binders such as methyl cellulose.

The present invention overcomes the limitations previous as it allows a porosity design not only in two but in the three directions of space through a mold previously designed and the mold is removed mechanically without need for heat treatment, chemical or any other type of treatment that can alter the composition of the piece. The Bioceramic piece is formed on the same mold by gelation of an aqueous suspension at room temperature without the need for apply pressure The presence of a binding agent does not a subsequent synthesizing treatment is necessary. This does also possible the incorporation of a biological substance or pharmacologically active to the initial composition of the ceramic, remaining this substance perfectly distributed throughout the structure. You get pieces of any bioceramics with interconnected pores in the three directions of space, with a appropriate hardness while easy to mold for use as implants for bone tissue regeneration.

Description Brief Description of the Invention

The proposed invention consists of a forming technique by which they can be prepared, at low temperature, bioceramics pieces with a porosity designed and interconnected in the three directions of space. The fact of working at these temperatures allows inclusion, during manufacturing process of substances biologically or pharmacologically active (drugs, hormones, bone growth factors, proteins, etc.) for later release as well as different cell populations

The method is based on solidification by gelation, at a temperature between 30 and 45 ° C, of a aqueous suspension of a ceramic (hydroxyapatite, phosphate tricalcium, bioglasses, siliceous mesoporous materials, etc. or combination of them) and a binder with characteristics of thermogel (agarose, gelane, etc. alone or mixed with substances such as gelatin, chitosan, alginates, dextrans). The consolidation is produces in a short time (less than 5 minutes), within a mold whose design determines the porosity of the piece due to the presence of a three-dimensional network of rigid filaments. The size of Pore, geometry and porosity percentage can be modified in the three directions of space.

The wet piece can be manipulated and cut Easily to get the desired shape. Also, the piece, which It contains a high percentage of water, it can be dried in an oven or be lyophilized, which allows its perfect preservation, which It is of great importance in the event that substances are introduced biologically or pharmacologically active. Lyophilization barely modify the dimensions of the pieces and allows a quick recovery of the original state in the presence of a fluid.

In addition to the advantages outlined (temperature of work below 45ºC, absence of aggressive solvents and versatility in the design of porosity and interconnection of pores) the proposed system is extraordinarily simple and economic.

Detailed description of the invention

First, the solution of the thermogel; for this a certain amount of product is weighed solid and inserted into a thermostated container containing an aqueous solution in continuous stirring. It increases temperature until melting / solubilization of the product, at which time the temperature of work and add pottery and other possible components. The working temperature depends on the thermal stability of the substances that are included in this stage. The system remains in stirring for a while to get a distribution homogeneous components. Finally, the suspension is poured into mold.

The mold basically consists of a Detachable parallelepiped that lacks top side, whereby The suspension is added. Two of the sides (hereinafter X and Y) are constituted by a base on which they have been fixed, perpendicularly, a series of rigid filaments (as a bed de fakir) according to the previously established design. The mold is design / manufacture in a way that ensures sufficient tightness during the consolidation process in both the X and Y dimensions as in the base of the mold (hereinafter Z).

Once the consolidation of the piece proceeds to remove the filaments of the dimensions X and And and the filaments are introduced along the third dimension Z, so that they pass through the intersections of the dimensions X and Y. After the process, the mold is removed and, in fresh, we proceed to the elaboration of the pieces with the form desired (cylinders, plates, cubes, etc.) by using Different types of cutting utensils.

Finally, the pieces obtained can be used wet or dry; The drying process can be performed by lyophilization or in an oven (at a temperature close to 37ºC), giving rise to porous pieces that will present different features.

The porosity of the pieces as well as the size of pore (between 300 and 1000 µm) will be determined by the Rigid filament dimensions, the number of filaments in each direction, its geometry and the drying process.

The advantages of working in the conditions of synthesis of the present invention (low temperatures and not use of aggressive or toxic solvents) are clear since allow, on the one hand the synthesis of three-dimensional scaffolding for tissue regeneration of bioceramics that cannot undergo heat treatment (eg carbonate hydroxyapatite nanoparticles precipitated at 40 ° C) and, on the other, the inclusion during the process of manufacture of biological or pharmacologically active molecules that Under these conditions they will not lose their activity. Also, during the manufacturing process can introduce other substances, such as polyethylene glycol, dextran, chitosan, polylactic acid, acid polyglycolic, etc., that interact with the active substance Controlling his release

The three-dimensional scaffolds synthesized in the The present invention can be used as supports for regeneration of different types of tissue, since all components used in the formulations tested have shown good biocompatibility and, in some cases biodegradability

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Brief description of the figures

Figure 1 (a) shows an outline of the top view of the mold with the filaments in the X and Y directions extracted while Figure 1 (b) shows a photograph from the top view of the mold where the filaments that can move on the X and Y axes.

Figure 2 (a) shows a diagram of the top view of the mold with the filaments in the X and Y directions inside, while Figure 2 (b) shows a photograph of the mold from a top view and with the filaments in the X and Y directions inside.

Figure 3 (a) represents a scheme of the introduction of the filaments in the Z direction within the mold, while Figure 3 (b) shows a photograph of This introduction in the mold.

Figure 4 shows different examples of parts prepared by the procedure described herein invention.

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Embodiment of the invention

The invention is illustrated by the following examples, which are not limiting of its scope.

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Example 1

The preparation of porous pieces of nanoparticles of carbonatehydroxyapatite and agarose, which includes Next steps:

Mold Preparation

Initially the fabric is designed and constructed three-dimensional to be used for the preparation of parts porous, determining the size of the mold and the dimensions of the rigid filaments In this case a cubic mold of 2 cm side and 1 mm stainless steel cylindrical filaments diameter.

Preparation of the suspension

Agarose powder is introduced into water inside of a glass reactor in a proportion of 2.5% weight / volume. Be raise the temperature of the suspension to 85 ° C, such that the agarose is melts, continuously stirring the suspension. The system cools and, when the suspension reaches 40 ° C, 10% weight / volume is added of the ceramic carbonatehydroxyapatite. The mixture is stirred for few minutes and poured into the designed mold. Suspension as well formed let stand a few minutes at room temperature, taking place the consolidation of it.

Preparation of the pieces

Once the paste is consolidated, the filaments of the X and Y directions are mechanically removed and the filaments are introduced along the third dimension so that they pass through the intersections of the X and Y directions. After a few minutes it is mechanically removed. the base containing the filaments along Z, obtaining a cubic block consisting of agarose / hydroxyapatite / water, with interconnected three-dimensional porosity. Visually, 1000 µm pores in the three directions can be seen in the wet piece. This piece can be easily cut with a blade, making cubes and other smaller parallelepipeds, cylinders,
etc.

The construction of cubic pieces was selected 0.5 cm side. These pieces were dried by lyophilization (freezing of wet product at -80ºC for 4 hours and later vacuum treatment for 24 hours). During the process the material loses all water, approximately 89%, suffers a slight contraction (less than 10%) and maintains its external shape. The pieces have a three-dimensional porosity with sizes of 900-950 µm pores, as well as others in size lower, between 0.5 and 100 µm, characteristic of the material and the drying process.

Characteristics of the pieces

The characterization of dry pieces by X-ray diffraction indicates the presence of diffraction maxima corresponding to hydroxyapatite and an amorphous background due to the agarose The FTIR spectrum indicates the presence of both components, not appreciating any interaction between them.

The material obtained behaves like a hydrogel. Thus, in the presence of an aqueous fluid, it immediately captures the fluid, swelling slightly, and recovering the initial behavior of an elastic and easily manageable material. This small swelling allows the perfect adjustment of the material to a bone defect when introduced as an implant in vivo and rehydrates when it comes into direct contact with the blood.

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Example 2

This example describes the incorporation of antibiotic vancomycin in porous bioceramic pieces of a sol-gel bioglass

Mold Preparation

As in example 1 the design and construction of the three-dimensional fabric that will be used for the preparation of porous pieces, determining mold size and dimensions of the rigid filaments. In this case a mold has been used 4 cm cubic side and stainless steel cylindrical filaments 1 mm in diameter.

Preparation of the suspension

First a mixture is prepared consisting of 6 grams of a sol-gel bioglass, of 70SiO2 / 26CaO / 4P2O5 molar composition and 0.4 grams of vancomycin

1 gram of agarose is introduced into 40 mL of water distilled inside a glass reactor. The temperature of the suspension at 85 ° C, until the agarose melts, stirring continuously. The system cools and, when the suspension reaches 40 ° C, the biovidrio / vancomycin mixture is added, Shake for a few minutes and pour into the designed mold. The suspension is allowed to stand a few minutes at room temperature, taking place the consolidation of it.

Preparation of the pieces

Once the pasta is consolidated they are removed mechanically the filaments of the X and Y directions and it introduce the filaments along the third dimension of so that they pass through the intersections of the X and e directions Y. After a few minutes, the base containing the base is mechanically removed the filaments along Z, obtaining a cubic block consisting of agarose / bioglass / vancomycin / water, with porosity three-dimensional interconnected. Visually, they can be seen in the wet piece pores of 1000 µm in all three directions. This Piece can be easily cut with a blade.

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The construction of cylindrical pieces of 13x4 mm was selected. These pieces were dried by lyophilization. During the process the material loses all the water, approximately 84%, undergoes a slight contraction (between 5 and 10%) while retaining its external shape. The pieces have a three-dimensional porosity with pore sizes of 900-
950 µm, as well as others of smaller size, between 0.5 and 100 µm, characteristic of the material and the drying process.

Characteristics of the pieces

The characterization of dry pieces by X-ray diffraction indicates that it is an amorphous material. He FTIR spectrum indicates the existence of the three components, not Appreciating no interaction between them.

When the pieces are introduced into a phosphate buffer solution at pH = 7.4 and at 37 ° C, the gradual release of vancomycin is observed by VIS-UV spectroscopy. Simultaneously to this process a swelling occurs due to the capture of the fluid by the hydrogel, so that the piece recovers its initial elasticity which allows its easy handling. This small swelling causes the piece to adjust to the bone defect when it is introduced as an implant in vivo when in direct contact with the blood.

Claims (14)

1. Method for preparing parts of Bioceramics comprising the following stages:

(to)

Prepare an aqueous suspension of a biocompatible ceramic in a binding agent with characteristics Thermogel

(b)

Design a framing mold three-dimensional filament based on porosity desired.

(C)

Insert the suspension into the mold.

(d)

Let consolidate the suspension in the gelation mold.

(and)

Mechanically remove the mold.

(F)

Remove the piece.

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2. Method for preparing parts bioceramics according to claim 1, wherein the ceramic is hydroxyapatite, tricalcium phosphate, a bioglass, material mesoporous siliceous base or combination of them. 3. Method for preparing parts bioceramics according to claim 1, wherein the polymer biodegradable is agarose or gelane, being able to mix with others substances such as gelatin, chitosan or alginates. 4. Method for preparing parts bioceramics according to claim 1, wherein the mold is a detachable three-dimensional structure based on rigid filaments constituted by an inert material and arranged in the three Space directions 5. Method for preparing parts bioceramics, according to claim 1, wherein the pieces are dried subsequently in an oven or by lyophilization. 6. Method for preparing parts bioceramics according to claim 1, wherein the solidification by gelation is performed at a temperature between 30 and 45 ° C 7. Method for preparing parts bioceramics, according to previous claims, where you can add biological substances to the aqueous suspension or pharmacologically active. 8. Method for preparing parts bioceramics, according to previous claims, wherein the Biologically or pharmacologically active substances are drugs, hormones, bone growth factors or proteins. 9. Method for preparing parts bioceramics, according to previous claims, wherein the suspension aqueous may contain substances that interact with the substance activates controlling its release as polyethylene glycol, dextran, Chitosan, polylactic acid or polyglycolic acid. 10. Bioceramics pieces obtained through of the claimed method with an interconnected and designed porosity in the three directions of space and even within it address with a pore size between 300 and 1000 \ mum. 11. Bioceramics pieces according to claim 10, which include one or more biologically active substances. 12. Bioceramics pieces, according to claims 10 and 11, including substances that facilitate the controlled release of the active substance. 13. Use of claimed bioceramics as implants for the regeneration of bone tissue. 14. Use of the claimed bioceramic pieces as a substance release system.