Bone substitute and method for producing the same

Abstract

The invention relates to a bone substitute (1) comprising A) a container (2) made of a porous casing (4) which is at least partly provided with openings; and B) a plurality of filler elements (5) which are not connected to one another and which are enclosed in the container (2); wherein C) the filler elements (5) consist of interconnected particles with an average diameter Dp; and D) the openings of the casing (4) are interconnected pores or channels with an average diameter of DM.

Description

The invention relates to a bone substitute, and to a method for producing the same.

Conventional production methods for producing bone substitute materials are known from the prior art which cannot completely control the porosity and also the geometry. Such production methods include, e.g., templating, emulsion methods, lost wax method. In particular, the geometry can be determined by these methods typically only in a limited range by the mold, for example. Thus, the outer edge of the geometry cannot be arbitrarily complex due to the required releasability. In particular, however, the internal geometry or architecture of the bone substitute material can be determined only via the porosity. It is limited by the geometry and arrangement of the pore fillers. Furthermore, for the formation of pores pore fillers are needed, which can lead to undesired interactions. Typically, only sphere-like pores can be produced. In addition to the limited pore geometry, the pore density or distribution within the bone substitute material is difficult to control. It would be desirable, therefore, to obtain a bone substitute, wherein the porosity specifically in the inner core of the supporting matrix or scaffold has a greater porosity than the edge region. Typically, however, it is just the opposite in the conventional methods, with the result that cell/scaffold interactions focus on the edge region.

The invention aims to remedy this situation. The object of the invention is to provide a bone substitute, which:

a) has a complex geometry with high porosity also in the interior;

(in the known methods of the prior art, the internal geometry is not freely controllable by the geometry and arrangement of pore fillers. In the conventional SFF methods (Solid Freeform Fabrication) such as 3DP and SLS, which are used for the method according to the invention channels having a geometry that cannot be chosen freely must be generated in the interior to enable removal of loose powder. The invention aims to remedy this situation and enables an interior architecture, which is not feasible with conventional SFF methods)b) allows the migration of cells;c) has an optimal surface to volume ratio, wherein per volume a surface as large as possible is available that is accessible for the bone cells;d) has a strength which allows the manipulation that is typically necessary of the bone substitute without damage;e) gets by with very little or no polymer components;(thus, there are no toxic components, e.g., remaining monomers and crosslinkers, there is no degradation during sterilization, problematic degradation products, such as, e.g., acids are avoided and there is no reduction in the rate of degradation.)f) enables a simple “depowdering” even of complex geometries inside the container through the freely moving filler elements andg) is simple and inexpensive to produce.

The invention solves this problem with a bone substitute as disclosed and claimed herein, and with a method for producing the same as disclosed and claimed herein.

Further advantageous embodiments of the invention can be commented as follows:

In a specific embodiment, the container has one or more windows passing through the casing. Thus, the loose particles can be removed from the cavity of the container through the window(s) by depowdering.

In another embodiment, the dimensions of the individual filler elements are defined by the longest enveloping circular cylinder of the diameter D_z.

In another embodiment, the average diameter D_P of the particles is between 1 μm and 250 μm.

In another embodiment, the average diameter D_M of the pores or channels is less than D_z. In another embodiment, each of the windows has a smallest dimension D_F which is governed by the formula D_Z>D_F>D_P.

In yet another embodiment, the diameter D_Z is larger than 200 μm, preferably larger than 500 μm.

In a further embodiment, the diameter D_F is larger than 50 μm.

In another embodiment, the filler elements and the casing are produced in one operation by means of a three-dimensional printing method, 3DP, or a selective laser-sintering method, SLS. Alternatively, the bone substitute according to the invention can be produced by producing a hollow cylinder, filling the cylinder with small particles and closing the ends of the cylinder with a hydraulic cement.

In another embodiment, the filler elements are spaced apart from one another, wherein preferably the minimal distance between the filler elements is larger than 50 μm. If the fillers are placed too close together, they can stick together.

In another embodiment, the casing contains, in addition to the openings and the windows, a number of passages having a diameter D_D which is at least equal in size to D_P and is at least 30 μm.

In a further embodiment, the casing has interparticle and intercrystalline interstices with an average diameter which is in the range of 0.1 D_P to 0.5 D_P, and is 1 to 50 μm.

In still another embodiment, the casing of the container and the filler elements are the reaction product of the solidification of a loose powder of the particles, preferably produced by means of 3DP or SLS.

EXAMPLES

• • a) solidification by crystal formation or polymerization (e.g., sugar or salt powder+water from the print head or as a further alternative, any powder+salt/sugar/polymer solution from the print head).
    • Example: sugar/CaP powder bed is sprayed locally with water droplets (from the print head via 3DP). Interlocking of the sugar crystals forms a matrix which holds together the CaP particles. Subsequent steps could include a further composite of CaP or washing out of the sugar crystals.
  • b) solidification by capillary forces: drying and interlocking of the surface of a powder;
  • c) solidification by gelation (e.g. alginate+Ca²⁺ ions=gel)
    • Example: cellulose powder is dissolved and solidified locally by a suitable printed solution;
  • d) solidification by cooling (e.g., a liquid medium is printed on a powder and solidifies by cooling);
    • Example: polymer solution printed on powder bed of any material.
  • e) solidification by sintering or melting and cooling (SLS or SLM (Selective Laser Melting) method).

In another embodiment, the casing of the container and the filler elements are different reaction products of the solidification of a loose powder of the particles, preferably produced by means of 3DP or SLS. The 3DP method allows for different solutions (e.g., with two print heads), which potentially lead to different reaction products. For example, by printing with water and phosphoric acid and an alpha-TCP, either CDHA (calcium-deficient hydroxyapatite) or DPD (monetite, dicalcium phosphate) can be produced.

In a further embodiment, the casing of the container and the filler elements are the reaction product of a hydraulic cement after its hardening, preferably produced by 3DP.

In yet another embodiment, the casing of the container and the filler elements are the melting product of the composite of a ceramic powder in an SLS process.

In another embodiment, the solidification is based on the reaction of one or more calcium phosphates or calcium sulfates with an aqueous solution.

In another embodiment, the calcium phosphate is the alpha-TCP, beta-TCP, hydroxyapatite, TetCP, or a mixture thereof.

In yet another embodiment, the final product contains DCPD, DCP or a mixture thereof.

In a further embodiment, the solidification takes place due to the reaction of

a) a powder mixture of MCP (Ca(H₂PO₄)₂) or MCPM (Ca(H₂PO₄)₂.H₂O) or a mixture thereof with

b) one or more of the following substances:

beta-TCP (Ca₃(PO₄)₂) or alpha-TCP (Ca₃(PO₄)₂) or hydroxyapatite (Ca₅(PO₄)₃OH) or calcium-deficient hydroxyapatite (Ca_10-x(HPO₄)_x(PO₄)_6-x(OH)_2-x, with 0≦x≦1) or tetra calcium phosphate (Ca₄(PO₄)₂O), and

c) an aqueous solution or pure water.

Additives to the aqueous liquid to be sprayed by the print head onto the powder bed are acids, hydrogels, alcohols, fats and surfactants. This provides the advantage that the acid required for the solidification reaction is made possible by the MCPM cement reaction with pure water. In doing so, significantly higher mechanical properties are obtained, since it is not the capillary forces that determine the local cement reaction rather a more homogeneous reaction is made possible by the powder layer and thereby a better connection between the individual layers of powder can be achieved.

In another embodiment, the porosity of the filling of the container defined by the filler elements is in the range of 1 to 70%, preferably 20 to 30%. This provides the advantage that the porosity present between the filler elements favors the formation of cells and blood vessels.

In another embodiment the filling elements have pores with an average diameter 1 μm to 50 μm.

In yet another embodiment, the casing also has any arbitrary, even non-rotationally symmetrical form, but the casing is formed preferably rotationally symmetrical.

In another embodiment, the casing has a thickness d>D_Z.

In another embodiment, the bone substitute comprises a plurality of interconnected containers, which are arranged side by side or one above the other.

In another embodiment, the bone substitute has, in addition to the plurality of filler elements which are not connected to one another, a number of interconnected filler elements.

In yet another embodiment, the casing has one or more column-like protrusions projecting into the interior of the container.

In a further embodiment of the container in the interior has one or more transverse beams, which are connected to the casing with both their ends.

In a specific embodiment of the method, the layers produced by the 3DP method or the SLS method have a thickness of 10 μm to 250 μm, preferably 30 μm-100 μm.

In a further embodiment of the method, the particles of the hydraulic cement forming the powder consist of MCP and MCPM and TCP and are hardened solely by means of water or aqueous solution. This provides the advantage that, unlike with other calcium phosphates, no phosphoric acid is necessary. Thus, the 3DP print heads (which are usually not designed for acids) are not only protected, but a high quality is made possible because the print head operates reproducibly. Furthermore, in this manner, the calcium/phosphate ratio can be accurately controlled.

In another embodiment of the method, the loose particles left behind inside the container by the 3DP method or the SLS method are removed through least one window from the container by applying one of the following techniques:

vacuum, rinsing with a medium in an ultrasonic bath or vibration.

This provides the advantage that the remaining particles of the powder which are not connected to one another form a kind of support function even for overhanging and nested geometries of the container and the filler elements in the 3DP and SLS methods. This is a crucial advantage over other rapid prototyping methods. Thus, the invention overcomes the disadvantage that the remaining particles must be removed. In this case the small movements of the filler elements and the resulting time-dependent deflection and turbulence of the air blown in enable depowdering even complex geometries in the interior of the casing.

A preferred use of the bone substitute is the in vivo colonization with body cells or in vitro culture of cell tissues.

Another preferred use of the bone substitute is the in vitro colonization of bone or cartilage cells.

Preferably, the bone substitute is used for filling or bridging of defects or cavities in bone. The invention and further developments of the invention are explained in more detail below by means of partly diagrammatic representations of several exemplary embodiments.

FIG. 1 shows a perspective view of an embodiment of the bone substitute according to the invention;

FIG. 2 shows a plan view onto the embodiment of the bone substitute according to the invention shown in FIG. 1;

FIG. 3 shows a longitudinal section through the embodiment of the bone substitute according to the invention shown in FIG. 1;

FIG. 4 shows a cross-section through the embodiment of the bone substitute according to the invention shown in FIG. 1;

FIG. 5 shows a side view of the embodiment of the bone substitute according to the invention shown in FIG. 1.

FIG. 6 shows a perspective view of a further embodiment of the bone substitute according to the invention;

FIG. 7 is a plan view of the embodiments of the bone substitute according to the invention shown in FIG. 6;

FIG. 8 shows a longitudinal section through the embodiments of the bone substitute according to the invention shown in FIG. 6;

FIG. 9 shows a cross section through the embodiment of the bone substitute according to the invention shown in FIG. 6; and

FIG. 10 shows a side view of the embodiment of the bone substitute according to the invention shown in FIG. 6.

FIGS. 1 to 5 show an embodiment of the bone substitute according to the invention 1 which essentially comprises a container 2 and a plurality of filler elements 5 which are enclosed in the container 2. The dimensions of the individual filler elements 5 are defined by their longest enveloping circular cylinder of diameter D_z. The filler elements 5 consist of interconnected particles with an average diameter D_P of at least 1 μm. The diameter D_Z of the longest circular cylinder enveloping a filler element 5 is larger than 200 μm. The bone substitute 1 may comprise a plurality of interconnected containers 2, which are arranged side by side or one above the other. Furthermore, the bone substitute 1, may comprise, in addition to the plurality of filler elements 5 which are not connected to one another, a number of interconnected filler elements 5.

The container 2 comprises a porous casing 4 which is at least partly provided with openings (not shown), which may have any shape but preferably is formed rotationally symmetrically (e.g., a hollow cylinder). The container 2 further has a container bottom 7, and a container top 8, wherein the container bottom 7 and the container top 8 may also be designed to be porous and at least partly with openings (not shown). The openings of the casing 4, of the container bottom 7 and of the container top 8 are interconnected pores or channels with an average diameter D_M, which is smaller than D_Z. Furthermore, the container 2 comprises a plurality of windows 6 passing through the casing 4, which are so dimensioned that no filler element 5 passes through one of the windows 6. For this purpose, the windows 6 have a smallest dimension D_F governed by the formula D_Z>D_F>D_P. The diameter D_F of the windows 6 is larger than about 50 μm.

Furthermore, the casing 4 may additionally comprise several passages 3 in addition to the openings and the windows 6 (FIGS. 6 and 8 to 10), the diameter D_D of which corresponds at least to the average diameter D_P of the particles forming the filler elements 5 to enable removal of the loose powder, but ideally is larger than 30 μm (angiogenesis). The casing 4 may comprise interparticle and intercrystalline interstices, which have an average diameter in the range of 0.1 D_P to 0.5 D_P, wherein the average diameter is between 1 and 50 μm. The thickness d of the casing 4 may be smaller than the diameter D_Z of the longest circular cylinders enveloping the filler elements 5. The casing 4 may have one or more column-like protrusions projecting into the interior of the container 2. In addition or alternatively, the container 2 can have one or more transverse beams, which are connected to the casing with both their ends. The porosity of the filling of the container 2 defined by filler elements 5 is in the range of 1 to 70%. The filler elements 5 are formed, for example, but not limited to, spherically and can have pores with an average diameter of 1 to 50 pm μm.

In addition to the conventional production methods, so-called SFF methods (Solid Free Form Fabrication) are known for producing bone substitute materials. In these so-called SFF methods the geometry is freely definable.

The present invention relates primarily to an application using the powder-based 3DP method (Three-Dimensional Printing) but also directly applicable to other powder-based SFF methods such as SLS (Selective Laser Sintering).

In the 3DP method, so-called 3D printers are used which include machines that build up three-dimensional work pieces. The work pieces are usually built under computer control from one or more liquid or powdered materials according to predetermined dimensions and shapes which can be defined by the CAD methods. The 3DP method is an additive method, wherein a work piece is produced by successively forming layers of material. In addition, physical or chemical hardening processes take place when building the work piece. For producing the work piece, the dimensions and shape are read by the machine and then the individual layers of liquid, powder, or plate-like material are successively deposited so that a work piece made of a series of cross-sectional layers is formed. To produce the work piece, these layers are automatically connected or fused together.

The SLS method is also an additive method, wherein three-dimensional structures are produced from a powdered starting material by sintering. The work piece is also built up layer by layer, wherein small particles of plastic, metal or ceramic are melted by means of high energy lasers (carbon dioxide laser). The material is selectively melted on the surface of a powder bed, so that a solid cross-sectional layer of the work piece is formed after hardening of the molten material. After a layer is complete, the powder bed is lowered by one layer thickness and a new layer of material is applied to the surface of the lowered powder bed. The process is repeated until the work piece is completed.

In one embodiment of the method according to the invention for producing the bone substitute 1, the casing 4 of the container 2 and the plurality of filler elements 5 are produced simultaneously in layers by means of 3DP or SLS methods. The layers produced by the 3DP or SLS method can have a thickness of 10 μm to 250 μm, preferably 30 μm-10 μm. Furthermore, the particles of the hydraulic cement forming the powder may consist of MCPM (Ca(H₂PO₄)₂.H₂O) or MCP (Ca(H₂PO₄)₂) and TCP and may be hardened solely by means of an aqueous solution or water. The loose particles left behind inside the container by the 3DP method or the SLS method and which have not hardened can be removed through at least one window 6 from the container 2 by applying one of the following techniques:

vacuum, rinsing with a medium in an ultrasonic bath or vibration.

Below, for the sake of simplicity, only the 3DP method is mentioned. However, this implies also alternative methods. Although the geometry is freely definable in these methods, undercut and overhanging and nested geometries must be supported, since the structure is built in layers. This in turn requires a later removal of these supports. In the case of 3DP and SLS the support function is taken over by loose powder, which has to be removed again later (depowdering). Nowadays, this is done normally with air pressure in the case of parts produced by 3DP and SLS. For this purpose, an airbrush nozzle is used, blowing filtered compressed air onto the bone substitute according to the invention. This will free from the outside the bone substitute produced according to the invention step by step from loose particles of the powder used. In particular, inside the bone substitute produced according to the invention this is done only with difficulties. Therefore, in SFF methods, the geometrical freedom in the interior is limited by the freedom of the removal of the free powder. If the remaining unhardened powder cannot be removed, the geometry is lost. In practice, this limits severely the choice of the geometry especially in the interior of a body. Typically, large channels have to be built, enabling a “depowdering”. The present invention focuses heavily on a better solution for the “depowdering” which thereby makes possible a new type of bone substitute. Moreover, this method allows for efficient and economical production.

Another inventive novelty relates to the composition of the powder. The prior art in 3DP for bone substitute is the following: An acid is applied onto the powder bed by means of a print head, whereby the ceramic powder particles are joined locally by means of a precipitation reaction. A novel method by mixing the ceramic particles (e.g., CaP calcium phosphates) with the particles which on contact with water form acid (e.g., MCP mono-calcium phosphate) allows printing with water. In addition to precise adjustment of the calcium/phosphate ratio this offers other advantages, described below in terms of the mechanical properties and the production method. One way to improve the mechanical properties of bone substitute according to the invention further lies in so-called “post-hardening” steps, i.e., the bone substitute produced according to the invention is, for example, enhanced by immersion in an acid bath, by thermal post-treatment, e.g., by sintering, or by chemical post-treatment (infiltration). In the case of the novel printing with a water-based and acid-free solution on a CaP/MCP powder bed, therefore, a new and completely unproblematic “post-hardening” is possible by solidifying the bone substitute at controlled or saturated humidity or by direct contact with water (with capillary soaking, immersing or spraying) by subsequent crystal formation.

The casing 4 of the container 2 and the filler elements 5 may be the reaction product of the solidification of a loose powder of the particles and are produced by means of 3DP or SLS. Examples include a) solidification by crystal formation or polymerization (e.g., sugar or salt powder+water from the print head or as a further alternative, any powder+salt/sugar/polymer solution from the print head (e.g., sugar/CaP powder bed is sprayed locally with water droplets (from the print head via 3DP). Interlocking of the sugar crystals forms a matrix which holds together the CaP particles. Subsequent steps could include a further composite of CaP or washing out of the sugar crystals; b) solidification by capillary forces: drying and interlocking of the surface of a powder; c) solidification by gelation (e.g. alginate+Ca²⁺ ions=gel); d) solidification by cooling (e.g., a liquid medium is printed on a powder and solidifies by cooling); or e) solidification by sintering or melting and cooling (SLS or SLM (Selective Laser Melting) method.

Alternatively, the casing 4 of the container 2 and the filler elements 5 may be the reaction product of a hydraulic cement after its hardening, preferably produced by 3DP, but also the reaction product of the composite of a, e.g., ceramic and polymer powder mixture in a SLS process.

Furthermore, the solidification may be based on the reaction of one or more calcium phosphates, or calcium sulfates with an aqueous solution. Here, the calcium phosphate may be alpha-TCP, beta-TCP, hydroxyapatite, TetCP, or a mixture thereof.

In alternative embodiments the solidification can take place due to the reaction of a) a powder mixture of MCP (Ca(H₂PO₄)₂) or MCPM (Ca(H₂PO₄)₂.H₂O) or a mixture thereof with b) one or more of the following substances: beta-TCP (Ca₃(PO₄)₂) or alpha-TCP (Ca₃(PO₄)₂) or hydroxyapatite (Ca₅(PO₄)₃OH) or calcium-deficient hydroxyapatite (Ca_10-x(HPO₄)_x(PO₄)_6-x(OH)_2-x, with 0≦x≦1) or tetra calcium phosphate (Ca₄(PO₄)₂O), and c) an aqueous solution or pure water.

The embodiment shown in FIGS. 6 to 10 of the bone substitute 1 according to the invention differs from the embodiment described in FIG. 1-5 only in that that the filler elements 5 are T-shaped or mushroom-shaped. Here, the filler elements 5 may comprise two essentially circular cylindrical portion, of which a respective first portion having a larger diameter than the adjoining second portion. Differently configured embodiments of the filler elements 5 are also possible and can be combined at will.

Although, as described above, various embodiments of the present invention are present, they are to be understood that the various features can be used both individually and in any combination.

This invention is therefore not just limited to the aforementioned particularly preferred embodiments.

Claims

1. A bone substitute comprising: a container made of a porous casing which is at least partly provided with openings; anda plurality of filler elements which are not connected to one another or to the container and which are fully enclosed within an interior of the container such that the plurality of filler elements cannot escape from the container;wherein individual filler elements are formed of interconnected particles, said particles having an average diameter DP,wherein the openings of the casing are interconnected pores or channels having an average diameter of DM,wherein the container containing the plurality of filler elements is a bone substitute,wherein the individual filler elements have a diameter DZ, said diameter being defined by a longest enveloping circular cylinder,wherein the container comprises at least one window passing through the casing, andwherein the at least one window has a smallest diameter DF which is governed by formula DZ>DF>DP.

2. The bone substitute according to claim 1, wherein the average diameter DP of the particles is between 1 μm and 250 μm.

3. The bone substitute according to claim 1, wherein the average diameter DM of the pores or channels is less than Dz.

4. The bone substitute according to claim 1, wherein the diameter DZ is larger than 200 μm.

5. The bone substitute according to claim 1, wherein the diameter DF is larger than 50 μm.

6. The bone substitute according to claim 1, wherein the filler elements and the casing are produced in one operation by means of a three-dimensional printing method or a selective laser-sintering process.

7. The bone substitute according to claim 1, wherein the filler elements are spaced apart from one another, and wherein a minimal distance between the filler elements is larger than 50 μm.

8. The bone substitute according to claim 1, wherein the casing contains, in addition to the openings and the at least one window, a number of passages having a diameter DD which is at least equal in size to DP and is at least 30 μm.

9. The bone substitute according to claim 1, wherein the casing has interparticle and intercrystalline interstices with an average diameter which is in a range of 0.1 DP to 0.5 DP, and is from 1 to 50 μm.

10. The bone substitute according to claim 1, wherein the casing of the container and the filler elements are a reaction product of a solidification of a loose powder of the particles.

11. The bone substitute according to claim 10, wherein the casing of the container and the filler elements are a reaction product of a hydraulic cement after hardening.

12. The bone substitute according to claim 10, wherein the casing of the container and the filler elements are a melting product of a composite of a ceramic powder in a selective laser-sintering process.

13. The bone substitute according to claim 10, wherein the solidification is based on a reaction of one or more calcium phosphates or calcium sulfates with an aqueous solution.

14. The bone substitute according to claim 13, wherein the one or more calcium phosphates is selected from the group consisting of alpha-TCP, beta-TCP, hydroxyapatite, TetCP, and mixtures thereof.

15. The bone substitute according to claim 10, wherein the solidification takes place due to the reaction of: a) a powder mixture of MCP (Ca(H2PO4)2) or MCPM (Ca(H2PO4)2.H2O) or both;b) one or more of beta-TCP (Ca3(PO4)2),alpha-TCP (Ca3(PO4)2),hydroxyapatite (Ca5(PO4)3OH),calcium-deficient hydroxyapatite (Ca10-x(HPO4)x(PO4)6-x(OH)2-x, with 0≦x≦1), andtetra calcium phosphate (Ca4(PO4)2O); andc) an aqueous solution or pure water.

16. The bone substitute according to claim 1, wherein the casing of the container and the filler elements are different reaction products of a solidification of a loose powder of the particles.

17. The bone substitute according to claim 1, the bone substitute contains DCPD, DCP or a mixture thereof.

18. The bone substitute according to claim 1, wherein a porosity of a filling of the container defined by the filler elements is in a range of 1 to 70%.

19. The bone substitute according to claim 1, wherein the filler elements have pores with an average diameter from 1 μm to 50 μm.

20. The bone substitute according to claim 1, wherein the casing is rotationally symmetrical.

21. The bone substitute according to claim 1, wherein the casing has a thickness d>DZ.

22. The bone substitute according to claim 1, wherein the bone substitute comprises a plurality of interconnected containers, which are arranged side by side or one above another.

23. The bone substitute according to claim 1, wherein the casing has one or more column-like protrusions projecting into the interior of the container.

24. The bone substitute according to claim 23, wherein the container in the interior has one or more transverse beams, which are connected to the casing at both ends.

25. A method for producing a bone substitute according to claim 1, wherein the casing of the container and the plurality of filler elements are produced simultaneously in layers by three-dimensional printing, selective laser-sintering or selective laser-sintering with ceramic.

26. The method according to claim 25, wherein layers produced by three-dimensional printing or by selective laser-sintering have a thickness of 10 μm to 250 μm.

27. The method of claim 25, wherein the filler elements are a reaction product of a hydraulic cement after hardening, and the hydraulic cement is formed of a powder consisting of MCP and MCPM and TCP and is hardened solely by means of water or an aqueous solution.

28. The method according to claim 25, wherein loose particles left behind inside the container by three-dimensional printing or by selective laser-sintering are removed through the at least one window from the container by vacuum, by rinsing with a medium in an ultrasonic bath, or by vibration.

29. The bone substitute according to claim 1, wherein there are no toxic components present in the bone substitute that would make it unsuitable for in vivo colonization with body cells or in vitro culture of cell tissues.