EP0717630A1

EP0717630A1 - Method of manufacturing implant materials

Info

Publication number: EP0717630A1
Application number: EP95922400A
Authority: EP
Inventors: Werner Mueller; Thomas Thaler
Original assignee: Sulzer Medizinaltechnik AG
Current assignee: Sulzer Medizinaltechnik AG
Priority date: 1994-07-08
Filing date: 1995-07-06
Publication date: 1996-06-26
Also published as: WO1996001641A1; AU2731295A; US5837235A

Abstract

According to the proposed method, implant materials with bone- and/or cartilage-regenerating properties are manufactured by reducing omentum or other fatty tissue from the patient's own body to small particles of tissue, preparing these particles in suspension form and transferring them to a carrier material by treating it with the suspension. This treatment can be in the form of, for example, filtration of the suspension through a powder-type or pre-shaped porous carrier material. The implant materials thus produced, which consist of carrier material and vital tissue cells embedded in it, are either in the form of a paste or pre-shaped, prosthesis-like implant materials, depending on the type of carrier material. The manufacturing process can be carried out in a closed apparatus, for example during an operation.

Description

METHOD FOR THE PRODUCTION OF IMPLANT MATERIALS

The invention relates to a method according to the preamble of the independent claim for the production of implant materials with bone and / or cartilage regenerating properties.

Healing of bone regressions, as can occur, for example, in the area of artificial prostheses, or also healing of difficult bone damage due to an accident can be brought about or improved or accelerated according to the prior art by using the body's own material, usually in the form, at the damaged areas a mixture of ground bone material and blood. The vital osteoblasts present in the implanted bone material initiate or support regeneration of bone material at the damaged site. Such bone material auto transplantations are very complex, but are considered the most promising way to bring about healing even in very difficult cases.

The main difficulty with autotransplantation of bone material lies in the fact that the removal of the material, for example from the , 9.

comb, is already a relatively difficult operation and that the amount that can be removed, especially in children, is very small. It can be attributed to these difficulties that the autotransplantation of bone material as a method with the highest chance of recovery is only used in the most difficult cases.

In order to overcome the difficulties of the quantitative availability of the body's own bone material, it is therefore proposed to remove it from the patient and then to increase the osteoblasts contained therein in vitro (AI Caplan, J. Orthop, Res. 9, 641-650, 1991 ). M.J. Doherty et al. (Bone and Mineral 25, Suppl. 1, page 9, 1994) have shown in rats that demineralized bone material implanted in the area of bone damage, which was previously coated with in vitro increased osteoblasts from rats, improved the healing of bone damage, namely more than implanted, demineralized bone material without such a coating, which also supports bone regeneration, but apparently cannot initiate it. The difference in the results of the two healing attempts was that when demineralized bone material was used alone, bone growth was observed only from the edges of the damage, that is to say from the living, damaged bone, while it was used on coated bone material thereon straight from the center of the damage. In many cases of treatment only with demineralized bone material, radio-ulnar waxes were observed, but none in the case of treatment with coated material.

Shigeyukui Wakitani et al. (The Jurnal of Bone and Joint Surgery, Vol. 76-A, No. 4, April 1994, pages 579 to 592), who treated rabbits with a collagen gel in order to treat defects in the stressed articular surfaces of knee joints, into which they introduced the body's own cells, obtained from bone marrow or periosteum and cultured in vitro. It turned out that the implant material particularly supported and improved the regeneration of the articular cartilage, but also the regeneration of the underlying bone. From the experiments described it can be deduced that cells derived from bone material (mesenchymal cells) are apparently capable, depending on the environment, of forming cartilage or bone.

The advantage of a method in which bone material is taken from the patient, multiplied in vitro and then implanted in the damaged area of a bone, compared to the method of direct autotransplantation, is that a significantly larger amount of vital material is available. The disadvantage is that an identical, not easy operation is necessary for the removal of the material, which also has to be separated from the implantation operation by, for example, six weeks for in vitro cultivation.

It is the object of the invention to show a method for producing an implant material with bone and / or cartilage regenerating properties, with which method the disadvantages of the known methods mentioned above, in particular the severity of the removal operation, the low, available amount of vital material or the temporal separation of removal surgery and implantation surgery can be avoided. In other words, this means that the method according to the invention is intended to make it possible for the body's own material, which is removed from the patient in an easy-to-perform removal operation immediately preceding the implantation operation, an implantation material. rial that can be inserted in the damaged area in an implantation operation immediately following the removal operation. When using an implantation material produced according to the method of the invention, the chances of healing should be significantly better than when implanting purely artificial (non-vital) materials.

The task relates to the technical problem of processing a suitable, body-own material between the removal operation and the implantation operation, that is to say in a period of approximately one hour, to form a suitable implant material. The advantages which result from the method according to the invention and from the implantation materials produced with the method according to the invention relate to the removal operation and to the chances of recovery, that is to say to the activity of the surgeon and to the patient's condition.

The technical problem is solved by the invention as defined in the claims.

The idea of the method according to the invention essentially consists in comminuting omentum or other adipose tissue removed from the patient into small particles, slurrying these tissue particles into a suspension, filtering the tissue particles out of the slurry, for example by filtering onto a carrier suitable for the damage to be treated apply material, whereby the suspension solution is separated. Depending on the implantation application, the carrier can be in powder or gel form and thus the finished implant material can be pasty, so that it is suitable for implants without requiring a mechanical strength function. net, for example for the treatment of missing bone and / or cartilage as a result of regressions, tumor removal etc. However, the carrier can also be more or less preformed and sponge-like or porous and consist, for example, of a ceramic or metallic material and depending on the material perform a mechanical strength function. It is also possible to apply the pasty material mentioned above to a carrier which takes over the mechanical function.

The removal of omentum or other adipose tissue is a very simple operation. In addition, such tissues can be removed in much larger quantities compared to bone material without harmful consequences for the patient. Between 50X10 ³ to 200X10 ³ vital cells can be isolated from one gram of adipose tissue, which is sufficient for the production of approximately one ml of paste-like implant material or for a corresponding volume of a porous shaped carrier.

In addition to other cells and non-cellular tissue parts, omentum or other fatty tissue also contains cells which are capable of forming ossified tissue and / or cartilage. This arises from the occurrence of ectopic bone injuries, which can occur under certain circumstances on body parts, on which no bone marrow and no bone skin are present, which are known to contain bone tissue and cartilage-binding osteoblasts or mesenchymal cells (see further above). Thus, if the tissue particles applied to the carrier material are exposed to a medium favoring bone or cartilage formation, they will initiate bone or cartilage formation in the same way as that by implanted, endogenous bone material with osteoblasts or osteoblasts cultivated in vitro on a carrier material or would do mesenchyma. In many cases, the surroundings of the steels to be treated will already suffice to create an appropriate milieu. Other means of producing a milieu favoring bone formation, such as, for example, bone matrix, hydroxyapatite, hydroxyapatite ceramic, etc., are known per se and need not be described in more detail at this level.

In addition to the cells that trigger the bone and / or cartilage tissue, adipose tissue also contains endothelial cells which, after implantation, lead to vascularization of the bone tissue that forms, as a result of which the tissue that forms is well supplied with blood and has a positive effect on the hay and mineral flush. Such cells may have to be removed for the regeneration of cartilage that is not supplied with blood.

Most of the non-vital bone implantation materials known per se come into question as carriers for implantation materials for bone defects, that is to say materials of biological origin (demineralized bone material, KoUagen sponge), degradable or non-degradable, synthetic polymers, mineral materials such as hydroxyapatite or hydroxyapatite ceramic or Metaüe, whereby, as already mentioned above, a part of these materials at the same time provide an environment which promotes bone formation. The carrier material can additionally be provided with growth factors such as TGFß (Transforrning Growth Factor Beta) or BMP (Bone Morphogenetic Protein) to promote mineralization. As already mentioned, the carrier can be in powder form and result in a pasty implant material, or it can be preformed and implanted to perform the strength function of the damaged bone or a part thereof. Metallic nets and plates, for example made of titanium, are particularly suitable for such functions. Carriers in the form of a sponge, in the form of fibrils, in the form of textiles (tissues, felts, etc.) or as a gel are suitable for implant materials for cartilage or bone surfaces covered with cartilage. Known materials of biological origin (eg KoUagen) and degradable and non-degradable, synthetic polymers can be used as carrier materials.

The detailed design of the process according to the invention is based on the design of the process for the manufacture of endoprostheses, which is described in European Patent Application No. 93810297.7 (publication number No. 0570331 AI) by the same applicant. It is essentially based on the following findings:

- The cells that initiate a bone flex need not be isolated from the vital tissue that is used for the production of the implant material. Other cells and non-cellular tissue parts do not interfere with the healing process. It is sufficient to comminute and suspend the tissue sufficiently fine to form tissue particles and to treat the carrier immediately therewith.

The tissue particles are produced by mechanical comminution of the tissue material removed and, if necessary, by a subsequent enzymatic digestion. Appropriate mechanical comminution ensures that the tissue particles formed have a narrow increase in size. As a result, the attack during enzymatic degradation becomes more regular and its product more homogeneous.

When using tissues with a very high fat content that could lead to fat embolism, it is advantageous to share by separating and separating the phases from the suspension.

The by-products resulting from the enzymatic digestion also do not necessarily have to be removed from the suspension before the

Carrier is treated with it by feeding. If necessary, the carrier can be rinsed with a rinsing solution for cleaning after the tissue particles have been stored, it also being possible to rinse out oily fractions. If the carrier is in the form of a gel, the tissue particles are separated from the suspension and taken up into the fermenting solution by counter-filtration, or the tissue particles are added and mixed before the gel completely solidifies.

Tissue particles with a size that is as uniform as possible and a high content of vital cells are obtained by precisely coordinating the mechanical comminution step and the enzymatic digestion step.

Of known enzymes, for example pancreatin, dispase, trypsin and others, Coüagenase has proven to be particularly suitable for digestion in in vitro experiments. Due to its specific effect, the Coüa¬ genase contributes to detaching individual witnesses, but also small witness associations, from the tissue without essentially damaging the vitality of the witnesses. If the collagenase had a hundred percent specificity for the coagulation of the extra-cellular matrix, it could possibly break down the tissue without damaging the cells or their surface in the least. This is an ideal picture insofar as it has been shown that cells that are exposed to the action of proteolytic enzymes, for example trypsin, for a prolonged period (for example two hours) damage theirs Show surface receptors, and that they can also completely void their vitality. Such effects are much weaker or not detectable with suitable CoUagenase preparations.

In other words, the exposure time of the enzyme must be long enough to split the tissue into sufficiently small particles, the time required for this very much depending on the size of the available particles. However, it must also not be too long in order not to unnecessarily reduce the quality of the extracted zeUen. Since for this reason there are no common optimal digestion parameters for differently sized ponds, appropriate mechanical comminution of the tissue must ensure that the tissue parts to be digested are of an optimal size for enzymatic digestion and are therefore approximately the same size as possible.

According to the invention, enzymatic digestion is preceded by such a gentle, mechanical comminution that tissue parts are created which are all as large as possible. Comminution processes are gentle on the material (atraumatic) if the tissue is cut and not squeezed or broken. According to the invention, this is achieved by treating the tissue with a plurality of very sharp, coordinated moving blades.

A narrow, precisely defined increase in size of the tissue particles to be digested allows a defined and optimized exposure time of the collagenase to be determined. The process according to the invention consists of the following process steps:

1. Production of a suspension of tissue particles from the body's own omentum or other fatty tissue,

2. (any) separation of excessively large tissue tissues and / or a large part of the fat portion,

3. (any) temporary storage of the suspension,

4. Applying the tissue particles to the carrier by feeding through the carrier (porous or powdery carrier), by feeding with the carrier (powdery carrier) by filtering and subsequent counter-filtering with a gelling liquid (gel-like carrier) or by mixing with another not completely solidified gel (gel-shaped carrier), 5th (possible) rinsing of the implant material consisting of carrier material and applied tissue particles,

6. (also) treatment of the implantation material to maintain the moisture necessary for the applied vital cells,

7. (Possible) interim storage of the implant material made.

Steps 1 and 4 are necessary, the remaining steps can be used or omitted, depending on the type of implant material to be produced.

In order to keep the traumatization of the cells as low as possible, the removed tissue is mechanically crushed very gently by treating it with a number of very sharp, coordinated, moving blades. The goal is to obtain the highest possible percentage of vital cells with a high absolute cell yield. Despite these requirements, comminution takes place in a relatively short time, for example 1-3 minutes / 100 g of tissue mass.

The size of the tissue that is mechanically shredded is limited by the size of the shredder to be used; typical input sizes are pieces with dimensions of 3x3x2 cm, 5x5x2 cm or, expressed in weight, pieces from 20g to about 50g. These pieces should be crushed into slices, sticks or cubes with dimensions of a few millimeters, preferably 1 to 3 mm, preferably 0.5 to 1 mm without subsequent digestion.

The mechanically comminuted tissue can then be digested enzymatically, for example with collagenase, in order to free the cells at least partially from the connective tissue and thereby further comminute the tissue particles. The collagenic and elastic parts of the connective tissue are broken down so that the cells are released but are damaged as little as possible (minimal breakdown of components of the surface of the block). The enzymatic breakdown should also take place as quickly (but gently) as possible, which is controlled via the temperature and the enzyme concentration. Moving the batch causes the phases to mix well, which further accelerates the breakdown.

Various such procedures for processing living cells are known and can be used accordingly. Surprisingly, it has been shown that stirring with a magnetic stirrer is more efficient than shaking the entire reaction vessel. Bodies without sharp edges, preferably rounded rods, are suitable as magnetic stirring bars, since they prevent the tissue from winding up. The stirrer is free floating or has only minimal contact with the ground, which also prevents grinding of the suspended cell material.

Coagagenase can be used for the digestion, for example, a recommended concentration is approximately 400 to 20,000 mandl U / ml, typically 750-30000 U / ml, the temperature at least 37 ° C. but not above 40 ° C. preferably 37-38 ° C and the volume ratio of tissue and enzyme solution 1: 1 to 1: 2.

After digestion, the suspension can be separated for partial purification. This part of the process must also be able to be carried out quickly since, on the one hand, one is dealing with material whose metabolic activity at processing temperatures is relatively high, in contrast to the processing of cooled tissue materials, and on the other hand, a process which can be carried out quickly for medical reasons is sought. Purification of the suspension with the aim of the quantitative separation of the enzymes used or the separation of cells other than those desired for bone and / or cartilage formation is, as already mentioned, not necessary for most applications.

Partial separation of the suspension to remove too large a part or part of the fat-containing part can be advantageous. This can either be carried out using the specific weight (letting settle and separating the lighter phase) and / or the size of the aggregates (feeding). Both separation methods are briefly discussed below. First the separation according to specific weight (letting the layers settle and separating): In the case of fatty tissues, the particles of the suspension which have a sufficiently large fat content float relatively quickly upwards. Contrary to human opinion, forced sedimentation by centrifugation is not necessary, but sedimentation in the gravitational field is sufficient. This means that the sedimentation can be carried out in a closed and dormant system.

The sedimentation can be accelerated, for example by a suitable choice of the depth of the sedimentation vessel, by dilution of the suspension after the enzymatic digestion has ended and / or by other suitable measures. Example: the suspension to be sedimented is diluted with buffer solution to 1.5 to 10 times, preferably to 1.5 to 2 times the volume, for example 300 g tissue + 300 ml enzyme solution = suspension for digestion (digestion ), which is made up to 1000 ml for sedimentation (which corresponds to a 1.67-fold dilution). In a cylindrical vessel 15 cm high and 10 cm in diameter, the sedimentation for these volumes takes 1-15 minutes, preferably 3-10 minutes. After this time, there is a clear boundary between the aqueous and oily phases. The sedimentation can be further supported by very slow stirring with a magnetic stirrer, since this frees lighter constituents which are enclosed in the sediment and, especially in the case of dense suspensions (CoUagenase: tissue = 1: 1), the separation of the tissue particles is promoted according to their specific weight by the additional weak movement.

Sedimentation and separation of the resulting layers is preferably carried out immediately after the enzymatic digestion. performed in the same vessel and at 20 - 40 ° C, preferably at 30 - 37 ° C for fatty tissue.

A separation of particles that are too large can be advantageous. In order to improve the cell yield and to prevent blockages, multi-stage sieves are recommended for larger quantities of material (from 50 g tissue). Up to 300g of fabric, for example 3 steps, the diameter of the sieves being 4cm - 6cm and the distance between two sieves being 2cm to 6cm. For the last sieve, a mesh size of 0.5 to 1 mm should be selected for exemplary, maximum particle dimensions from 0.5 to 1 mm 0.25 mm. This avoids that the entire mass of the remaining fraction loads a single sieve layer more and more, which increases increasing flow resistance and, above all, drastically changes the sieve effect, so that the larger the load, the larger it is the passing Teü¬ chen decreases, whereby considerable losses of Zeümaterial occur. In the case of multi-stage sieves, on the other hand, the total quantity, the number and the mesh size of the sieves used are distributed accordingly and the flow resistance and the sieve effect are influenced much less.

The sieves are preferably arranged horizontally one above the other in order to be able to carry out the feeding by means of gravity, which enables a more uniform loading of the sieve and thus in turn a more defined feeding. With such an arrangement, fewer devices are also required, which in turn also complies with the strict sterility requirements.

However, feeding by pump transport is also possible, for which essentially the same criteria must be observed. A pump transport has the main advantage that it prevents filtration gravity can be carried out, which enables air-free liquid transport in a simple manner, which is important for the subsequent treatment of the carrier material.

Peristaltic pumps are used as pumps, preferably those with 2-roller heads. Surprisingly, it has been shown that this results in only insignificant losses of vital cells. In contrast, the use of such pumps enables a controlled, not simply subjected to the force of gravity in a closed system, which is not only of linerical importance with regard to sterility, but also for the time-controlled implementation of the method.

Dissolved components of the suspension, such as the CoUagenase, which are used for the enzymatic degradation, and liquid components, such as oily fat from the tissue, especially if only a separation by size has been carried out, are only later at / after the Treatment of the carrier material removed.

The suspension is then filtered through the powdery or porous and preformed, if necessary appropriately moistened carrier material. If it is a powdered carrier material, it can also be added to the suspension and, together with it, filtered through a suitable filter or at least partially separated from the suspension solution by being allowed to settle. In such a case, it may prove to be advantageous to also add an agent promoting the adhesion such as fibronectin to the suspension. In the form of a gel-like carrier, the suspension is filtered through a correspondingly fine feed and the tissue particles are thereby separated from the suspension solution. Immediately afterwards, a leaching liquid (for example cold, newly rinsed CoUagen solution) is pressed through the feed in the opposite direction and the tissue particles are thus taken up in the liquid, which is then left to stand for gelation.

In order to meet the high sterility requirements of the method, the suspension must be kept in a closed system that also contains the carrier during the entire process, preferably including application to the carrier material. This is made possible by, for example, hose connections between the digestive vessel and the coating module, with which the cell material is stored in the carrier material. The liquid can be transported by means of gravity (different heights in the arrangement of the devices) or by means of peristaltic pumps, roller pumps or other displacement pumps, preferably those in which the suspension comes into contact only with disposable parts. In the following example, a Watson pump with 2 rollers with a silicone tube of approx. 8 mm diameter and without slippage was used for the liquid delivery to apply the cell material to a carrier material. It has been shown that the number of vital cells in the suspension is not significantly reduced even by pumping five times.

So that the quality of the implant material produced is preserved until use, it can be specially protected as the last step of the method until wound closure. The vital witnesses are thus protected from drying out. Subsequently, a sketchy example of a closed device for carrying out the method according to the invention is discussed with the aid of the figure. The individual device components are, in some cases, those that are available on the market. The interconnection of these devices in accordance with the new, compressed and, surprisingly, biologically very effective method described here, on the other hand, corresponds to the process sequence according to the invention and thus represents a device for the production of implantation materials according to the patent claim. The device can be designed such that it can also be can be used in an operating room and enables the production of implantation material according to the invention on site.

The figure shows, in a schematically simplified manner, a completely lockable device in which the entire method can be carried out in a coherent, closed vessel and hose system. It can be easily arranged on a mobile frame and is thus mobile for use, for example, in the laboratory or in the operating room. The visual coherence naturally does not exclude the supply of tissue, reagents and solutions to be processed. However, the unity should show how one can meet the very strict sterility requirements.

A first unit comprises the devices for mechanical comminution, a drive 1, a conveying section 3, which is directed towards a pair of knives / perforated plates 5, 7, and a digestive cell 9 with a stirrer 10 for support to disintegrate the tissue material to release the cells digestion and a first sedimentation. The devices can be switched so that the shredded tissue mass immediately falls from the perforated plate 7 into the digestive vessel. After completing the digestion this can The sedimentation also takes place in order to separate the oily phase. To this end, the vessel can be filled with buffer at the start of sedimentation to support the separation. Furthermore, a slight movement of 0.1-1 U / sec can be generated with the same aim, for example with a magnetic stirrer. The suspension formed in the first unit is conveyed by a conveying means 13 into a second unit, which consists of a feed unit 15 and a collecting vessel 17. During the conveyance into the vessel 17, a slight flow is also generated in the vessel 9. The feed unit 15 here is a multi-stage filter for the extraction of particles of no particular size, which are collected as a suspension in the collecting vessel. In this form, the sedimentation tendency of the suspension is only slight and is canceled in the vessel 17 by a second magnetic stirrer 18. Another conveying means 19 transfers the ready-to-use, homogeneous suspension into the third unit 25 for feeding onto the carrier material. This unit consists of a container in which the, for example, powdery carrier material is introduced in such a way that the incoming suspension flows through it evenly. It may be advantageous to condition the dry carrier material with a physiological solution before filtering the suspension.

A powdered carrier material can also be supplied to the vessel 17 and filtered through a feed integrated in the unit 25 or can be removed directly from the vessel 17.

The filter 15 can also be connected without a vessel 17 between the vessel 9 and the unit 25 for treating the carrier material, in such a way that the suspension is guided by the feeder directly onto the carrier material without intermediate storage. In the case of a gel-shaped carrier, which is produced by fermentation of a liquid into which the tissue particles have been taken up, at least one feed that is sufficiently fine for the filtration of the tissue particles from the suspension desired for the implantation material is used, which Feed rate is removed from the suspension after filtration and a gelling liquid is pumped through the feed in the opposite direction. Above the feed, the gelling solution with the tissue particles contained therein is removed from the vessel (line 20 indicated by dash-dotted lines) and guided into the vessel 25 serving as a gelling vessel in this case, where it gels, for example, with appropriate heating.

Depending on the size and complexity of the overall device, it can be equipped for the series production of implant materials or for the ad hoc production of individual portions of such materials in the operating room.

Claims

P A T E N T A N S P R Ü C H E

Process for the production of implantation materials with bone and / or cartilage-regenerating properties by applying vital cells or tissue associations from the body's own tissue to a carrier material, characterized in that omentum or other fat tissue is used as the body's own tissue, for the at least partial liberation of tissues and cell structures from the tissue this is comminuted into tissue particles, that a suspension is produced from the tissue particles and that a carrier material is treated with the suspension.

2. The method according to claim 1, characterized in that the carrier material is powdery, spongy, textile-like or preformed and porous and that the treatment of the carrier material consists of a filtration of the suspension through the carrier material.

3. The method according to claim 1, characterized in that the carrier material is powdery and that the treatment of the powdered carrier material consists of adding the carrier material to the suspension and subsequently allowing it to settle or feed.

4. The method according to Anspmch 3, characterized in that the suspension is added an agent promoting cell adhesion or that such an agent is present on the carrier material to be treated.

5. The method according to claim 1, characterized in that the carrier material is gel-shaped and that the treatment of the carrier material consists of filtering the tissue particles from the suspension and taking up the tissue particles in a gelling liquid.

6. The method according to any one of claims 1 to 5, characterized in that the tissue particles are made by mechanical comminution of the tissue.

7. The method according to any one of claims 1 to 5, characterized in that the suspension is produced by mechanical comminution of the tissue to pieces and subsequent enzymatic digestion of the tissue pieces to particles of the desired particle size.

8. The method according to any one of claims 5 or 7, characterized in that the mechanical comminution is realized by a plurality of sharp blades moved in correlated fashion.

9. The method according to any one of claims 1 to 8, characterized in that before the treatment of the carrier material by filtration too large particles are separated from the suspension.

10. The method according to any one of claims 1 to 8, characterized in that before the treatment of the carrier material by letting settle and separating the lighter layer of fat-resistant materials are separated from the suspension.

11. The method according to any one of claims 1 to 10, characterized in that Trägπnaterial and ^' by treatment with the suspension applied to it tissue particles are treated with a rinsing solution.

12. The method according to any one of claims 1 to 11, characterized in that a material of biological origin, a metallic material, a minerüsches material or a degradable or non-degradable, artificial polymer or textile is used as the carrier material.

13. The method according to any one of claims 1 to 12, characterized in that the carrier material is provided with growth factors to favor mineralization.