ES2378044B2 - MANUFACTURE OF THREE-DIMENSIONAL ANDAMIOS WITH MESOPOROUS BIOACTIVE GLASSES BY FAST PROTOTIPATE. - Google Patents

Abstract

Manufacture of three-dimensional scaffolding with bioactive mesoporous glasses by rapid prototyping. # A macroporous scaffolding (200 - 1,000 microns) is manufactured from a mesoporous bioactive glass of previously known porosity (2 - 50 nm) that is mixed with the necessary components to obtain a paste of the consistency necessary so that it can be injected by a rapid prototyping robot. The subsequent elimination of these adjuvants by calcination leads to the obtaining of a pure and manageable ceramic material. # The method allows to preserve this initial porosity and thus obtain a scaffold of three-dimensional porosity designed and interconnected. In addition, the scaffold does not have polymers in its composition, thus avoiding the possible toxicity problems that may arise.The composition and structure of the scaffolds manufactured make them optimal for use in tissue engineering for bone regeneration and as a release system for active principles.

Description

Manufacture of three-dimensional scaffolding with bioactive mesoporous glass by rapid prototyping.

Technical sector

The present invention falls within the technical field of manufacturing materials for bone tissue regeneration. More specifically, the invention relates to the manufacture of macroporous scaffolds of controlled and orderly three-dimensional porosity, through the rapid prototyping technique. The scaffolds obtained are usable in tissue engineering.

State of the art

Tissue engineering consists of sowing and adhesion in vivo of human cells on a structure, a scaffold. The cells, once implanted, proliferate, migrate and differentiate into specific tissue while secreting the necessary components to create the required tissue. The choice of the structure to be implanted is crucial so that it allows the cells to behave in a suitable way to produce the tissues of a certain shape and size. Various materials have been used to manufacture these structures or scaffolds for tissue engineering, both ceramic materials and synthetic and natural polymers derived from calcium phosphates and collagen.

Among these materials, bioactive glasses are a family of materials known and used in clinic for the regeneration of small bone defects for decades. These materials have the ability to form a stable bond with the surrounding tissue (the bone) once they are implanted in the body.

The recent inclusion of mesoporosity in bioactive glasses has meant a significant advance in these materials. On the one hand, the increase in the specific surface implies a greater chemical reactivity, so the kinetics of the chemical processes involved in the bioactive process are significantly greater. On the other hand, uniform porosity and high pore volume give these materials the ability to act as "carriers" of drugs (López-Noriega. A. et al. Chemistry of Materials 2006, 18, 13, 3137).

Bioactive mesoporous glasses, which have already demonstrated their biocompatibility in cellular assays (Mayor, M. et al. Acta Biomaterialia 2009, DOI: 10.1016 / j.actbio. 2009.09.008), have characteristics that make them excellent candidates for being used as scaffolding components for tissue engineering for bone regeneration. Its rapid bioactive kinetics guarantees a stable and prompt union of the scaffold with the bone tissue. The possibility of incorporating high doses of antibiotics or anti-inflammatories into these materials could be used to reduce the complications inherent to the surgical intervention of the implant such as infection processes or inflammatory processes,

or to promote the regeneration of bone tissue (loading antiostreoporotic drugs, bone morphogenic protein, ...). On the other hand, it is known that the dissolution products of bioactive glasses favor the proliferation of osteoblasts (bone-forming cells), so that the in vivo bone regeneration capacity would be even more favored.

Therefore, in addition to the importance of its composition, scaffolds for tissue engineering must meet a series of structural requirements that guarantee the correct proliferation of cultured cells, osteoblasts. The pores must have a size of 300-500 microns that allow the correct fixation of the cells in the material. In addition, these must be interconnected by windows that allow cell migration and a correct perfusion of fluids for nutrition and cellular respiration.

In order to achieve these objectives, macroporous three-dimensional scaffolds of bioactive mesoporous glass have already been synthesized (Y. Zhu et al. Microporous and Mesoporous Materials 2008, 112, 494; X. Li et al. Chemistry of Materials 2007, 19, 4322; H. Yun et al Journal of Biomedical Materials Research Part B: Applied Biomaterials 2008, 374). Most of the techniques used to manufacture these scaffolds with these materials are based on the introduction and subsequent elimination of polymeric templates during the synthesis of bioactive mesoporous glasses. However, the use of such templates (polyurethane foams, methyl cellulose) lead to the obtaining of materials with poor mechanical properties and without any macroscopic morphological control that guarantees the interconnection of the macropores of the scaffold.

The rapid prototyping technique has been used to overcome the inconvenience of using templates in the synthesis of macroporous scaffolds. This technique allows the manufacture of materials with the desired structure from a design previously made in CAD format. In rapid prototyping a robot-injector reproduces the scheme of the piece to be manufactured previously programmed by injecting a paste in which the material of which the scaffolding will be composed is located. This ink is a stable suspension of the material together with possible adjuvants necessary to make this suspension in the appropriate solvent. The subsequent evaporation of the solvent from this paste results in the scaffolding. This technique allows to manufacture shaped materials, macroporosity and controlled interconnections, since they have been previously designed.

There are documents that describe the manufacture of macroporous scaffolds of bioactive mesoporous glass by rapid prototyping (H. Yun et al Chemical Communications 2007, 2139, H. Yun et al. Chemistry of Materials 2007, 19, 6363. US2008 / 0103227) where they are obtained scaffolding composed of a bioactive mesoporous glass polycaprolactone composite.

The manufacturing method is based on a synthesis intermediate, a precursor to glass, from which a porous material is formed using a polymeric template, which is subsequently removed by calcination, in order to generate a porous material (1-100 nm). The final macroporisity of the scaffolding is achieved by adding a polymer (such as polycaprolactone) to the porous material obtained once ground, and then rapid prototyping. The materials that are obtained have a very small specific surface which entails a lower chemical reactivity and therefore a reduced bioactive kinetics. In addition, the presence of a polymer implies a series of associated problems. For example, PGLA (polyglycolic-polylactic acid) is rapidly degraded, saturating the body's ability to remove it from tissues. On the contrary, other polymers such as ε-PCL present serious problems to be reabsorbed as a result of their biostability. The scaffolds thus obtained have poor mechanical resistance in addition to presenting a defective mesoporous order, which prevents control of the final surface and porosity values of the materials.

In view of the state of the art, it would be desirable to develop a scaffolding manufacturing method that allows obtaining materials of known, orderly and controlled porosity, which confers on textural properties (speci fi c surface, pore volume) and improved bioactives for use as implants in tissue engineering.

For this, the present invention proposes a method of manufacturing scaffolding where it is based on a bioactive material of known porosity that does not undergo modifications in such porosity in the scaffolding manufacturing process. During such a process, the porosity of the initial material is preserved while achieving a desired macroporosity using the rapid prototyping technique. A macroporous scaffold of pure mesorporous bioactive glass is thus obtained, which does not require the presence of a biopolymer thus achieving a better bioactive kinetics and allowing the scaffold to be used as a carrier of large doses of a drug. The technique can also be applied to form any mesoporous silica material such as SBA-15 or MCM-41.

Brief Description of the Invention

A method for obtaining three-dimensional macroporous scaffolds of highly ordered mesoporous bioglass is described. Starting from pure mesoporous glass, three-dimensional scaffolds with pre-designed porosity are prepared both at the mesopore (2-50 nm) and macroporo (200-1,000 microns). In this way, specific scaffolds can be prepared for each bone defect, whose properties are determined before three-dimensional printing. These properties allow, in the first place, the bone colonization of the implant and the reabsorption of the material to transform it into neoformed bone.

The method consists of grinding a biactive mesoporous glass of known porosity. Once ground, the powdered glass is suspended in an organic solvent (dichloromethane or chloroform) and mixed with a solution of PCL in the same solvent. The solution thus obtained is stabilized, the solvent being evaporated until a paste of adequate consistency is obtained for rapid prototyping and obtaining the scaffolding. The scaffold obtained is dried and the PCL is removed by calcination.

Throughout the process the textural and structural characteristics of the starting material do not vary significantly and the chemical composition remains stable, so that materials with a rapid bioactive kinetics and a highly ordered mesoporous structure are obtained that allow it to be used as a carrier of large doses of active principles and confers the ability to act as a liberating system for such active principles.

This process presents a series of advantages that improve the techniques developed so far. First, obtaining a mechanically stable material without a polymer in its composition avoids the inconvenience of its presence, such as its degradation in the form of potentially toxic compounds. Secondly, the starting compound is a mesoporous material whose textural and structural characteristics are not modified during the manufacturing process, and therefore the process allows obtaining a highly ordered mesoporous material. Third, the use of rapid prototyping leads to the manufacture of materials with known and controlled morphology and interconnected macroporosity. Finally, the technique described in the present invention would be foreseeably useful for any mesoporous material of siliceous base, so it opens the possibility of obtaining macroporous materials with different mesoporous characteristics (in terms of arrangement, diameter and pore volume).

Detailed description of the invention

The method starts from a bioactive mesopore glass of known pore size to which it is subjected to different stages to reach a scaffold of the same pure mesoporous glass, with the same pore size as the starting glass, and of known macroporosity. Mesoporous glass of known porosity, in turn, can be previously synthesized, following the procedure detailed in the literature (Chem. Mater, 2006, 18 (13), pp. 3137-3144). The proposed method steps are:

1-Grinding in anhydrous organic medium of the synthesized glass until a particle size of less than 32 microns is obtained.

2-Suspension of the powdered glass in dichloromethane and mixed with a solution of polycaprolactone in dichloromethane.

3-Ultrasonic stabilization of the resulting solution. Evaporation of the solvent until obtaining a paste with the consistency necessary to perform a correct deposition with the prototyping machine.

4-Injection of the paste by means of a rapid prototyping robot. Scaffolding drying.

5-Elimination of polycaprolactone by calcining the scaffold at 800 ° C for 3 hours.

The glass obtained can be characterized by various techniques to ensure that it is a highly ordered mesoporous material (Figure 1) (Figure 2), macroporous (Figure 3) and highly bioactive (Figure 4).

Brief description of the fi gures

Figure 1 shows the N2 adsorption isotherms of the bioactive mesoporous glass scaffold, where the relative pressure (Pr) is plotted against the amount of adsorbed N2 (Ca). The hysteresis cycle is characteristic of materials with open mesopores at both ends. The Figure also includes the pore size distribution (Vp), which shows a pore diameter (Dp) of about 5 nm.

Figure 2 shows the low-angle X-ray diffractogram of the mesoporous glass scaffold. Low angle diffraction maxima indicate the presence of a highly ordered mesoporous structure (cubic ordering in this case).

Figure 3 shows the distribution of macropore by mercury intrusion. It can be seen that after calcination (continuous line) the materials have a macroporosity of around 300 microns, in addition to the macroporosity of the scaffold (500 microns).

Figure 4 shows the infrared spectra with Fourier transform of the scaffold before and 3 days after carrying out an in vitro bioactivity test in SBF (simulated body fluid acronym). It can be seen that after 3 days of immersion a new layer of crystalline calcium phosphate, a marker of the bioactivity of the material, has appeared on the surface of the material.

Embodiment of the invention

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

Example

The preparation of a macroporous scaffold of mesoporous bioactive glass is described.

Preparation of mesoporous glass of known pore size

2 g of P123 are dissolved in 30 g of ethanol with 0.5 g of 0.5 N HCl. Then 3.7 g of tetraethylorthosilicate (TEOS), 0.34 g of triethyl phosphate (TEP) and 0.49 g are added. of Ca (NO3) 2.4H2O at intervals of three hours under continuous agitation. After 12 hours, the soles obtained are deposited in Petri dishes. The geli fi cation takes place after 35 hours of evaporation of the solvents. The gels are allowed to age for a week after which they are removed in the form of transparent membranes (less than 0.5 mm thick). Both the surfactant and nitrates are removed by a heat treatment in air at 700 ° C for 3 hours, obtaining the glass in powder form, with a pore size of 5 nm, as the final product.

Grinding of synthesized mesoporous glass

4 g of mesoporous glass thus synthesized are taken and ground in a 1 cm diameter agate ball mill with 100 ml of ethanol for 1 hour. The material obtained is screened below 32 microns.

Obtaining mesoporous glass paste for rapid prototyping

4 g of ground and sieved glass are mixed with 100 ml of dichloromethane and dispersed using ultrasound. To this dispersion is added 100 ml of another solution of 2.7 g of high density polycaprolactone in dichloromethane. The resulting mixture is allowed to evaporate until a paste is obtained with the consistency necessary to be injected by the rapid prototyping machine.

Obtaining scaffolding by rapid prototyping

The prototyping machine allows the manufacturing of previously designed structures through CAD programming. In the example detailed here the pieces obtained dimensions of 1 cm x 1 cm x 1 cm.

Scaffolding characterization

The N2 adsorption isotherms of the bioactive mesoporous glass scaffold (Figure 1) show a characteristic hysteresis cycle of materials with open mesopores at both ends and also show the pore size distribution, with a pore diameter of about 5 nm.

The low-angle X-ray diffractogram of the mesoporous glass scaffold (Figure 2) presents the low-angle diffraction maxima that indicate the presence of a highly ordered mesoporous structure (cubic ordering in this case).

Figure 3 shows the distribution of macropore by mercury intrusion. It is observed that the materials have a macroporosity of around 300 microns, in addition to the macroporosity of the scaffold (500 microns).

The infrared spectra with Fourier transform of the scaffold before and 3 after carrying out an in vitro bioactivity test in SBF (Figure 3) indicate that after 3 days of immersion a new phosphate layer has appeared on the surface of the material crystalline calcium, a marker of the bioactivity of the material.

Claims (10)

1. Method for the preparation of three-dimensional macroporous scaffolds of bioactive mesoporous glass comprising the following steps:

(to)

Grinding in anhydrous organic medium of a bioactive mesoporous glass of known porosity (2-50 nm).

(b)

Suspension of the pulverized glass in an organic solvent and mixing with a solution of polycaprolactone in the same solvent.

(C)

Stabilization of the resulting solution and evaporation of the solvent to obtain a paste.

(d)

Injection of the paste obtained in a rapid prototyping robot and subsequent drying of the obtained piece.

(and)

Elimination of polycaprolactone by calcination.

2.

Method for the preparation of three-dimensional macroporous scaffolds of bioactive mesoporous glass, according to claim 1, where the grinding is carried out in anhydrous organic medium until a particle size of less than 32 microns is reached.

3.

Method for the preparation of three-dimensional macroporous scaffolds of bioactive mesoporous glass, according to claim 1, wherein the ground glass is suspended in dichloromethane or chloroform and mixed with a solution of polycaprolactone in the same solvent.

Four.

Method for the preparation of three-dimensional macroporous scaffolds of bioactive mesoporous glass, according to claim 1, wherein the stabilization of the solution is carried out by ultrasound.

5.

Method for the preparation of three-dimensional macroporous scaffolds of bioactive mesoporous glass, according to claim 1, wherein the calcination of the scaffold is carried out at 800 ° C for 3 hours.

6.

Three-dimensional macroporous scaffolds of bioactive mesoporous glass obtainable through the claimed method characterized in that they have a pre-designed and interconnected porosity of both mesoporo (2-50 nm) and macroporo (200-1,000 microns).

7.

Three-dimensional macroporous scaffolds of bioactive mesoporous glass, according to claim 6, which may include active ingredients.

8.

Use of the three-dimensional macroporous scaffolds of bioactive mesoporous glass claimed as implants for the regeneration of bone tissue.

9.

Use of the three-dimensional macroporous scaffolds of bioactive mesoporous glass claimed as an active ingredient release system.

SPANISH OFFICE OF THE PATENTS AND BRAND Application no .: 201000353 SPAIN Date of submission of the application: 03.17.2010 Priority Date: REPORT ON THE STATE OF THE TECHNIQUE 51 Int. Cl.: See Additional Sheet RELEVANT DOCUMENTS

Category

56 Documents cited Claims Affected

TO

YUN, HUI-SUK, et al .; Three-dimensional mesoporous-giantporous inorganic / organic composite scaffolds for tissue engineering; Chemistry of Materials, 2007, volume 19, No. 26, pages 6363-6366. 1-9

TO

YUN, HUI-SUK, et al .; Fabrication of hierarchically porous bioactive glass ceramics; Key Engineering Materials, 2008, volume 361-363, pages 285-288; ISSN 1013-9826. 1-9

TO

US 2008103227 A1 (YUN, HUI-SUK, et al.) 01.05.2008, paragraphs [0014] - [0015]; Examples 1-3. 1-9

TO

YUN, HUI-SUK et al .; Design and preparation of bioactive glasses with hierarchical pore networks; Chem. Commun., 2007, pages 2139-3141. 1-9

Category of the documents cited X: of particular relevance Y: of particular relevance combined with other / s of the same category A: reflects the state of the art O: refers to unwritten disclosure P: published between the priority date and the date of priority submission of the application E: previous document, but published after the date of submission of the application

This report has been prepared • for all claims • for claims no:

Date of realization of the report 22.03.2012

Examiner N. Vera Gutierrez Page 1/4

REPORT OF THE STATE OF THE TECHNIQUE Application number: 201000353 CLASSIFICATION OBJECT OF THE APPLICATION A61F2 / 28 (2006.01) A61L27 / 10 (2006.01) B29C67 / 00 (2006.01) Minimum documentation sought (classification system followed by classification symbols) A61F, A61L, B29C Electronic databases consulted during the search (name of the database and, if possible, terms of search used) INVENES, EPODOC, CAS, WPI, EMBASE, BIOSIS, MEDLINE, NPL State of the Art Report Page 2/4  WRITTEN OPINION Application number: 201000353 Date of Written Opinion: 22.03.2012 Statement

Novelty (Art. 6.1 LP 11/1986)

Claims Claims 1-9 IF NOT

Inventive activity (Art. 8.1 LP11 / 1986)

Claims Claims 1-9 IF NOT

The application is considered to comply with the industrial application requirement. This requirement was evaluated during the formal and technical examination phase of the application (Article 31.2 Law 11/1986).  Opinion Base.- This opinion has been made on the basis of the patent application as published. State of the Art Report Page 3/4  WRITTEN OPINION Application number: 201000353 1. Documents considered.- The documents belonging to the state of the art taken into consideration for the realization of this opinion are listed below.

Document

Publication or Identification Number publication date

D01

YUN, HUI-SUK, et al .; Chemistry of Materials, 2007, volume 19, No. 26, pages 6363-6366. 2007

D02

YUN, HUI-SUK, et al .; Key Engineering Materials, 2008, volume 361-363, pages 285-288; ISSN 1013-9826. 2008

D03

US 2008103227 A1 01.05.2008

2. Statement motivated according to articles 29.6 and 29.7 of the Regulations for the execution of Law 11/1986, of March 20, on Patents on novelty and inventive activity; quotes and explanations in support of this statement The invention relates to a method for the preparation of macroporous three-dimensional scaffolds of bioactive mesoporous glass comprising the following steps: a) grinding in anhydrous organic medium of a bioactive mesoporous glass of known porosity; b) suspension of the pulverized glass in an organic solvent and mixing with a solution of polycaprolactone in the same solvent; c) stabilization of the resulting solution and evaporation of the solvent to obtain a paste; d) injection of the paste into a rapid prototyping robot and subsequent drying of the piece obtained; e) elimination of polycaprolactone by calcination. Document D01 discloses the preparation of three-dimensional scaffolding for tissue engineering from bioactive mesoporous glasses. The synthesis process begins with the spraying of a bioactive mesoporous glass, selecting the size fraction smaller than 25 micrometers. A solution of polycaprolactone in chloroform is then prepared to which the pulverized glass is added, thus forming a homogeneous paste that is subjected to extrusion by means of a rapid prototyping robot. Document D02 describes a porous bioactive glass useful in the preparation of three-dimensional scaffolds for tissue regeneration and controlled drug release. The material is obtained by mixing a precursor solution of glass with methylcellulose, forming a paste that is stabilized by ultrasound and subjected to rapid prototyping and subsequent calcination at 600 ° C. In document D03 a porous material with hierarchical pore structure useful in the manufacture of scaffolds for tissue engineering is described. Two types of materials are prepared, depending on whether they present 2 or 3 types of pores. The material with 2 types of pores (example 1) is obtained by mixing the powdered mesoporous glass (less than or equal to 50 micrometers) with chloroform and polycaprolactone until a uniform paste is obtained, which will be injected into a rapid prototyping robot. In order to obtain the material with 3 types of pores (examples 2 and 3), it is based on a precursor solution of the porous material to which a structure directing agent is added. The paste obtained, homogenized by ultrasound, is extruded by means of a rapid prototyping robot and subsequently dried and calcined at 600-1000 ° C for 4 hours. In view of documents D01-D03, it is considered that the invention as defined in claims 1-9 of the application meets the requirements of novelty and inventive activity according to Articles 6.1 and 8.1 of the Patent Law. State of the Art Report Page 4/4