EP3810216A1

EP3810216A1 - Bioactive substrate

Info

Publication number: EP3810216A1
Application number: EP19729515.7A
Authority: EP
Inventors: Armin Lenhart; Lukas Forchheimer; Gundula Schulze-Tanzil; Clemens Gögele
Original assignee: Klinikum Nuernberg Medical School GmbH; Georg Simon Ohm Hochschule fuer Angewandte Wissenschaften Fachhochschule Nurnberg
Current assignee: Klinikum Nuernberg Medical School GmbH; Georg Simon Ohm Hochschule fuer Angewandte Wissenschaften Fachhochschule Nurnberg
Priority date: 2018-06-21
Filing date: 2019-06-06
Publication date: 2021-04-28
Also published as: WO2019243066A1; DE102018114946B3

Abstract

The invention relates to a bioactive substrate for the colonization of living cells, comprising a porous three-dimensional glass scaffold, which has an alkaline earth metal oxide content of not more than 2.0% by weight and whose alkali metal oxide content is reduced at least on its surface by leaching.

Description

Bioactive carrier

The invention relates to a bioactive carrier for the settlement of living cells.

Osteoarthritis is a common joint disease that is economically very important due to the treatment costs and incapacity to work, the importance of which is increasing against the background of the current demographic change. It is currently incurable, marked by a

progressive course, whereby, among other things, the cartilage is attacked in the joints, loses its strength and is partially dissolved. Osteoarthritis often arises as a result of articular cartilage injuries because the articular cartilage and cartilage tissue in general have no self-healing ability. As a treatment option for the reconstruction of the articular surfaces after

Cartilage injuries and advanced osteoarthritis often remain

appropriate surgical intervention, which is often accompanied by a partial or complete replacement of the joint with an implant. Alternatively, the possibility of cultivating the body's own cartilage tissue in vitro is sought as a biological replacement. After replacement, this replacement tissue is transferred to the patient's cartilage defect in order to replace the defective articular cartilage.

To ensure a three-dimensional arrangement of the cartilage cells, natural and synthetic biomaterials are used, which change as soon as the

New tissue formation in the defect has progressed, should resolve.

This process of obtaining tissue by culturing cultivated cells in a suitable biomaterial is called "tissue engineering". This means that cell proliferation of adherent cells and subsequent tissue formation on a three-dimensional bioactive carrier, usually called "scaffold", is achieved in vitro. In order to achieve this in vitro growth of chondrocytes, so-called chondrocytes, cells taken from the body have to be stimulated in a suitable environment. A suitable geometrical arrangement of the cells can form the new one Support tissue. For the in vitro growth of the cells and a

Tissue formation is of major importance:

There must be a suitable scaffold for the growth of the cells, i.e. a bioactive carrier that not only meets the mechanical requirements placed on such a carrier in future tissue, but is also sufficiently cyto- and biocompatible to do this To ensure cell survival and not trigger inflammation.

Living and reproducible cells must be able to be settled on the bioactive carrier, the so-called scaffold. That is, a sufficient

Cell adhesion to the scaffold must be possible. The cell surface receptors must therefore recognize structures in the scaffold as binding motifs and the necessary cofactors (including ions) must be sufficiently available.

Furthermore, excitation and control of a cell type-specific

Signal transduction of the living cells may be possible, which can be achieved, for example, by growth stimulators (such as growth factors, ions, but also a suitable scaffold topology).

Finally, for an initial cell proliferation, that is, a cell multiplication and a cell growth, as well as a later differentiation of the cells, there must be a beneficial extracellular milieu in the scaffold and around the cells.

Of particular interest is the the geometric scaffold structure (topology) and growth factors-controlled synthesis of a new one

extracellular cartilage matrix through the cells in the scaffold, since the articular cartilage consists of over 90% extracellular matrix (ECM = extracellular matrix). With the stimulation factors for cell growth and for matrix synthesis, one can essentially differentiate between electro / biochemical, structure-associated and mechanical, factors. If the colonized cells are to multiply well, these factors should be selected accordingly for the cell type to be grown. In the context of tissue engineering, the bioactive carrier, i.e. the scaffold, is a central element, because on the one hand it must be mechanically sufficiently stable, on the other hand it must enable cell adhesion, cell growth and the synthesis of extracellular matrix (ECM synthesis). It also has to be resolvable, whereby the concentration gradients of ions with corresponding chemical and electrochemical potentials that result from the dissolution of the bioactive scaffold cause cell proliferation

Can support cell differentiation and ECM synthesis. In this context, glass is regarded as an excellent material for the production of such a bioactive carrier.

Bioactive carriers, also called bio glasses, are known in various applications, for example from WO 2011/161 422 A1 or WO 2016/089 731 A1, furthermore from WO 2014/168 631 A1, which are also explicitly concerned with the production of a scaffold. However, the bioactive carriers or bio glasses described there are based on alkaline earth and phosphate

Glass systems that form a hydroxyapatite layer on the glass surface in aqueous solutions. This hydroxylapatite layer forms an interface, as a so-called interface, to which the living cells to be multiplied must adhere and which should promote growth. The best known of these

Organic glasses containing alkaline earth are known under the designation 45 S5.

The problem with these organic glasses, however, is that the hydroxyapatite layer acts as a diffusion barrier, which at least hinders, if not prevents, the subsequent dissolution of the framework-like glass support in the body.

There is also the risk that the hydroxyapatite layer influences cell differentiation. Because hydroxyapatite forms the basis of

Hard substance of the bones and teeth of animals and humans. This can lead to the cell type of the applied living cells changing during cell growth, that is to say that a cartilage cell differentiates into a bone cell as a result of the presence of the hydroxyapatite. Therefore, it is not

it is ensured that the hydroxyapatite layer growth or

is differentiation-neutral. The invention is therefore based on the problem of specifying an improved bioactive carrier. To solve this problem, a bioactive carrier for the settlement of living cells is provided according to the invention, with a porous three-dimensional glass framework which has an alkaline earth metal oxide content of at most 2.0% by mass and which is at least superficially reduced in its alkali metal oxide content by leaching ,

The bioactive glass carrier according to the invention is characterized by a porous three-dimensional web structure which has a specific glass composition with regard to the content of alkaline earth metal oxides and alkali metal oxides. According to the invention, an extremely low content of alkaline earth metal oxides is contained, the content is a maximum of 2.0% based on the dry carrier, ie the pure dry substance without embedded OH groups and

Water molecules, preferably less than 1.5% by mass, in particular less than 0.5% by mass. The glass composition is characterized in that there is no addition of any alkaline earth metal oxide.

This has the particular advantage that hydroxylapatite layer formation is avoided in any case. This means that the three-dimensional glass bridge structure has no hydroxylapatite on the surface. In addition, the content of alkali metal oxides in the bioactive carrier according to the invention is also reduced by targeted leaching via an acidic surface treatment. Alkali metal oxides are in the

The basic composition of the glass from which the bioactive carrier is made is generally available to a sufficient extent, since these alkali oxides, usually Na2Ü and K2O, serve as network converters and are required to adjust the melting and sintering temperature. After making the

three-dimensional bridge structure, these alkali metal oxides are deliberately removed from the glass matrix. This means that the Na ⁺ and K ⁺ ions from the Glass matrix detached by the acid attack, i.e. released from its bond in the network. There is therefore an ion exchange between Na ⁺ and K ⁺ in the network and H ⁺ in the glass. This leads to a drained gel layer with a minimal amount of alkali.

A first advantage of this leaching or ion exchange is that when this glass is stored in aqueous solutions (cell culture medium) there are no dissolving reactions of the glass, in which the pH value changes significantly into the toxic range. This applies in particular to the local area on the surface of the glass, this is the contact area between the adherent cells and the glass.

A second advantage of this leaching i.e. The ion exchange is that OH groups or water can be stored in this “open” network. This inclusion and the ion composition on the scaffold surface have proven to be advantageous for the adhesion of the cells and the stimulation of the cell receptors that interact with the surface of the scaffold.

The bioactive carrier according to the invention therefore allows very good adhesion and thus colonization with the living cells to be multiplied, in particular cartilage cells. Furthermore, the bioactive carrier according to the invention shows no discernible tendency that the cell differentiation is not chondrogenic i.e. in a differentiation direction than z. B. to influence cartilage cells, as is the case for known carriers with the hydroxyapatite layer. This means that the bioactive carrier behaves neutrally with regard to cell differentiation.

Due to the high proportion of alkali oxides in the base glass, the leaching and the non-bridging oxygen or OH bonds, the corresponding assemblies within the glass matrix are only slightly linked as described. This leads to a reduced chemical resistance and promotes the dissolution of the carrier when used in vivo, which means that the

Glass carrier also dissolves due to the hydrolytic acid-alkali attack of the surrounding medium given an in vivo use. As described, one of the central features of the bioactive carrier or bioglass according to the invention is that a superficial strong one

There is a reduction in alkali metal oxides due to leaching. This

Alkali metal oxide reduction can only be superficial. This means that the thin webs are only reduced in the area of a relatively narrow surface layer. As an alternative to this, the alkali metal oxide content can also be reduced far in depth, even to the extent that the bridge framework is reduced in alkali over the entire bridge cross section.

The reduction, i.e. leaching, takes place in 1 normal

Hydrochloric acid, for example at 37 ° C instead. As described, the ion exchange between Na ⁺ / K ⁺ and H ⁺ takes place here, as does the incorporation of H2O or OH- into the glass, so that a glass matrix with a high water or OH group content is formed. So water is stored, similar to a gel layer. A superficially reduced alkali metal oxide content can be achieved after only about 1 to 1.5 days for leaching. A complete ion exchange of a scaffold bar can be achieved after approx. 12 days, whereby a bar thickness between 30 - 120 pm is required.

The alkali metal oxide content should be reduced as far as possible. The content of alkali metal oxides in the leached-out area of the finished bioactive carrier should be a maximum of 6.5% by mass (based on the dry substance), be it superficial or over the entire cross-section, preferably it should be less than 4% by mass. The degree of leaching is naturally greater the longer the leaching takes place.

On the growth process or the matrix synthesis and the

In addition to the three-dimensional geometry of the support, the tissue formation process through the cells to be multiplied also influences the concrete geometry of the bar framework. The pore size of the bridge scaffold should be between 20 and 300 pm, in particular between 50 and 200 pm. This means that the pores are sufficiently small so that they can contain cells and ECM formed by them can be fully populated or closed within a few days or weeks.

The web thickness should be between 30 and 120 mm, in particular between 50 and 100 μm. This is expedient in that, as described, the bioactive carrier, that is to say the glass bar structure, is dissolved during later in vivo use. The thinner the webs, the faster the resolution.

As described, the glass composition is essential for the bioactive carrier according to the invention. The following mass percentages relate to the dried carrier, i.e. to the pure dry substance. According to the invention, the bioactive carrier (in mass%) contains:

- Si0 ₂ 77.5 - 85.0

- P2O5 8.5 - 12.0

- B2O3 0.5 - 3.4

- Na ₂ 0 + K2O <6.5

As already stated, the bioactive carrier according to the invention is distinguished by the fact that it has only a minimal content of alkaline earth metal oxides, in particular CaO, which, as described, is disadvantageous

Hydroxyapatite layer formation leads. As stated, the content of alkaline earth metal oxides, in particular CaO, is a maximum of 2.0% by mass (based on the dry substance), preferably significantly less. The measurement of the respective amounts or proportions takes place, for. B. with X-ray fluorescence or

Flame Spectroscopy ICP.

In more specific terms, the bioactive carrier can contain (in mass% based on the dry substance):

- Si0 ₂ 79.5 - 83.0

- P2O5 9.0 - 11.7

- B2O3 0.65 - 3.25

- Na ₂ + K2O <6.5 Furthermore, the bioactive carrier can additionally contain (in mass% based on the dry substance): - Ti0 ₂ 0.1 - 0.5 and / or

- Zr0 ₂ 0.1 - 0.4

Furthermore, the bioactive carrier can additionally contain (in mass% based on the dry substance):

- CuO max. 0.1 and / or

- ZnO max. 0.1 and / or

- NaCI max. 0.5 and / or

- NaF max. 0.4 and / or

- Na ₂ S0 ₄ max. 0.6 and / or

- KNOs max. 0.6

In addition to the bioactive carrier according to the invention, the invention further relates to a specific glass composition according to the invention which is suitable for the

Production of a bio glass, in particular just such a bioactive carrier, as described above, was formulated. This invention

Glass composition contains (in mass%):

- Si0 ₂ 60 - 65.5

- P ₂ 0 ₅ 6.7 - 9.3

- B ₂ 0 ₃ 0.4-2.6

- Na ₂ 0 25.5 - 28.5

- K ₂ 0 0.45 - 1.55

and an alkaline earth metal oxide content of <2% by mass.

The content of alkaline earth metal oxides in the invention

Glass composition is less than 2% by mass, especially less than 1.5% by mass, preferably less than 1% by mass and in particular less than 0.5% by mass.

The glass composition according to the invention and thus also the bioglass that can be produced essentially contains the network formers S1O _2, P ₂ 0s and B ₂ O3 and the network converters Na ₂ Ü and K ₂ O. Because of the proportion of P ₂ O ₅ and B ₂ O3 it is possible to set the Si0 ₂ content between 60.0-65.5 mass%, it preferably being in the range between 62-63 mass%. Due to the addition of P ₂ O ₅ , the glass contains the phosphate ions which are necessary for cell proliferation and are usually present in the cell culture medium anyway and essentially buffer the pH. B ₂ O ₃ serves to stabilize the glass structure, the boron ions also proving to be beneficial in terms of cell proliferation.

The network formers Na2Ü and K2O are to the specified proportions

required for melting. The higher the proportion, the thinner the glass becomes and the chemical resistance decreases. The proportion of Na2Ü and K2O was chosen as high as possible for the glass composition according to the invention, since the leachability increases with the alkali fraction and the remaining residual glass is only slightly polymerized. This is advantageous for in vivo resolution. The selected composition is also advantageous in that there is a high diffusion coefficient on the part of the alkali, that is to say the Na ⁺ and the K ⁺ , which has a very beneficial effect on the high and rapid leachability. As described, it is possible to leach out the surface of the glass web superficially or completely, so that the alkali metal oxide content is <6.5% by mass, preferably <4% by mass, based on the dry matter.

In more specific terms, the glass composition can contain (in mass%):

- Si0 ₂ 61, 5 - 64.0

- P2O5 7.0 - 9.0

- B2O3 0.5 - 2.5 - Na ₂ 0 26.0 - 28.0

- K ₂ 0 0.5 - 1, 5, whereby here too the proportion of alkaline earth metal oxides is as low as possible, that is <2 mass% or significantly less.

In a further embodiment, the glass composition according to the invention can additionally contain (in mass%): T1O2 0.1-0.5 and / or

- Zr0 ₂ 0.1 - 0.4

T1O2 and / or Zr0 ₂ can be added to promote or increase the stability within the leached layer. As a result of the leaching, there are slight tensions within the matrix due to the solid Si bonds. After the finished bioactive carrier is stored in a liquid medium, usually a TRIS buffer, these do not have a negative effect. The gel layer can be stabilized by adding T1O2 respectively

Zr0 ₂ can be achieved because the Ti and Zr ions form permanent bonds.

In a further embodiment it can be provided that the glass composition additionally contains according to the invention (in mass%):

- CuO max. 0.04 and / or

- ZnO max. 0.1

There is also the possibility of CuO and / or ZnO

Add glass composition. Copper ions have an antibacterial effect, zinc ions have an anti-inflammatory effect. This means that the addition of minimal amounts of CuO and / or ZnO can also have a positive effect on cell growth. It is also possible to additionally add to the glass composition according to the invention (in mass%): - NaCl max. 0.5 and / or

- NaF max. 0.4

These compounds can be used to introduce fluoride and / or chloride ions into the glass matrix. These have a favorable influence on the proliferation of the cells, which is why their addition is advantageous even with a small proportion.

It is further provided in a further embodiment that the glass composition according to the invention additionally contains (in mass%): - Na ₂ S0 ₄ max. 0.6 and / or

- KNO3 max. 0.6

These two compounds are preferably added to the batch in order to homogenize and refine the melt.

At this point the information that the stated mass% values refer to the glass composition of the respective components of the raw material mixture before melting. The mass% values on the molten glass can be recorded in a corresponding manner with the corresponding values. All that is varied is the proportion of Na2Ü and K2O that are leached out as alkali metal oxides from the finished bioglass carrier, as described in the introduction. In the event that the melted glass is to be used as bioglass in a non-leached form, the corresponding Na2Ü and K ₂ 0 proportions are also found on the melted glass.

A concrete glass composition contains (in mass%):

- Si0 ₂ 62.7 - P2O5 7.0

- B2O3 2.0

- Na ₂ 0 25.6

- K ₂ 0 1.0

- Ti0 ₂ 0.12

- Zr0 ₂ 0.1

- CuO 0.02

- ZnO 0.06

- NaCI 0.3

- NaF 0.2

- Na ₂ S0 ₄ 0.4

- KN03 0.5

At this point, it is indicated that, although a specific composition has been described above, any composition that results in combination of the chemical components within the specified intervals is essential to the invention. That means that any numerical value in 0.01 - steps within the respective, to a chemical component

specified interval is to be considered as essential to the invention and therefore any of these numerical values, although not explicitly described, for

Specification of an interval or a proportion of a chemical component can be used.

The invention further relates to a method for producing a bioactive

Carrier of the type described above, with the following steps:

Use of a glass composition of the type described above and manufacture of a glass melt therefrom

Production of a glass body from the glass melt

Grinding the glass body to form a glass powder

- Formation of a suspension from the glass powder and an aqueous or organic solvent Application of the suspension to a porous sponge carrier made of a polymer while covering the surface of the sponge carrier with the glass powder

Heat treatment of the coated sponge carrier for thermal

Decomposition of the sponge carrier and simultaneous sintering of the

Glass powder for the formation of a porous three-dimensional frame structure Leaching of the frame structure to reduce the content

Alkali metal oxides in the bridge framework

To produce the bioactive carrier according to the invention, a

glass composition according to the invention, as described above, used. This means that the corresponding chemical constituents in the corresponding amount according to the invention, as they result from the

corresponding interval information or quantity information result, are mixed. This glass composition is melted in the next step, that is to say that a glass melt is produced, from which a

Vitreous is poured. For this, the high-purity, powdery

Glass composition after appropriate weighing and optionally homogenization, preferably in a ceramic mortar, melted in a platinum-gold crucible, such a crucible being used to avoid melting contamination. The melting preferably takes place at approx. 1,300 ° C, the holding time is selected depending on the corresponding glass composition, it is approx. 5-7 hours.

The melt, which has a viscosity of approx. 10 ² dPas, is then poured into a preheated steel mold, removed from the mold and transferred in solid form to another furnace in order to relieve tension during the further cooling. Any casting geometry can be created here.

Then the vitreous is crushed and ground. This can be done in a suitable mill, for example a ball mill. The grinding is carried out until a correspondingly fine glass powder is obtained, an appropriate grain size fraction being separated off if necessary. The glass powder is then mixed with an aqueous or organic solvent to form a suspension, which is then further processed to form the carrier.

For this purpose, a porous sponge carrier made of a polymer is used according to the invention. This sponge carrier, which has the desired geometry of the bioactive carrier to be produced, serves as a carrier for the ground glass powder. For this purpose, a suspension is applied, which the glass powder is applied to the porous sponge carrier, so that its surface is covered with the glass powder. That means that the powder particles on the

Adhere to the sponge surface, thus the sponge geometry is reproduced over the glass powder. The coating with the glass powder takes place in such a way that the most uniform possible particulate coating is on the

Surface forms.

In the next step, the occupied is heat treated

Sponge carrier. This serves two purposes, firstly the thermal decomposition of the sponge carrier, which means that it is thermally destroyed; on the other hand, the simultaneous sintering of the glass powder to form a porous three-dimensional framework as the basis for the bioactive carrier. By transferring the polymer sponge carrier, which is degraded by the heat treatment with regard to its organic components, and the simultaneous sintering of the powder particles to one another, despite the dissolution of the

Sponge carrier formed a stable, porous, three-dimensional web structure. It should be noted here that the glass particles are sintered, which means that the glass particles adhere to one another by viscous flow without crystallizing. The web structure formed shows the sponge structure reduced in proportion.

In the last step, the three-dimensional bar framework produced in this way is then leached out in order to reduce the alkali metal oxide content. The finished bioactive carrier or the scaffold is then stored in a fluid, preferably a TRIS buffer, until it is used for cell growth.

The glass powder itself can be sieved as described, a glass powder fraction with a grain size <100 mm, in particular <40 μm, is preferably formed, with which the suspension is subsequently produced.

The suspension can preferably be prepared with isopropanol as the solvent, although other solvents can also be used.

Furthermore, a dispersant and / or a binder can be added to the suspension. The dispersant serves to avoid the formation of agglomerates, while the binder, preferably polyvinyl alcohol, has an emulsifying effect.

A sponge made of polyurethane is preferably used as the sponge carrier, for example one from Eurofoam Deutschland GmbH under the

Brand name "Bluebrain S" distributed reticulated polyurethane foam based on polyester.

The heat treatment for the degeneration of the polymer sponge carrier and for the sintering of the glass particles takes place at a temperature of 550-800 ° C, in particular 600-740 ° C. This temperature is sufficient to decompose the organic sponge and at the same time to sinter the glass particles together. The sintering time can range from a few minutes to 2 hours.

The finished sintered glass bridge framework is leached out with hydrochloric acid, preferably 0.1 molar hydrochloric acid, preferably at a slightly higher level

Room temperature, preferably about 37 ° C. Leaching takes place depending on the size of the scaffold or the web diameter and the desired degree of leaching or the desired depth of leaching. On

Superficial ion exchange of Na ⁺ / K ⁺ from the glass matrix and H ⁺ from the leaching medium is possible within approx. 1-2 days, a complete one Ion exchange across the web diameter, with a web width of approx. 80 - 100 miti, can be achieved in approx. 12 days of leaching.

Further advantages and details of the present invention result from the exemplary embodiments described below and from the drawings:

1 shows a bioactive carrier with a porous three-dimensional glass framework before the leaching of the alkali metal oxides,

Fig. 2 shows the bioactive carrier from Fig. 1 after the leaching of

Alkali metal oxides,

Fig. 3 is a diagram for explaining the essential steps of the

Process according to the invention for the production of a bioactive carrier according to the invention,

4 shows a scanning electron microscope image of the fracture surface of a web of a leached bioactive carrier,

Fig. 5 is a diagram of a line scan over the fracture surface of the

leached web from FIG. 9 along the scan line drawn in FIG. 9, showing the courses of the element concentrations for silicon ions and sodium ions,

6 shows a microscopic image of living porcine articular chondrocytes on a bioactive carrier according to the invention after 24 hours

Colonization, FIG. 7 shows a microscopic image of living porcine articular chondrocytes on a bioactive carrier according to the invention after 21 days of colonization, 8 shows a microscopic picture of living human mesenchymal stromal cells on a bioactive carrier according to the invention after 24 hours of colonization, FIG. 9 shows a microscopic picture of living human mesenchymal painters

Stromal cells on a bioactive carrier according to the invention after 7 days of colonization,

10 shows a microscopic image of living human fluff fibroblasts on a bioactive carrier according to the invention after 24 hours

Settlement, and

11 shows a microscopic image of living human fluff fibroblasts on a bioactive carrier according to the invention after 7 days of colonization. 1 shows a microscopically enlarged image of a bioactive carrier, that is to say a bioglass scaffold, before the alkali metal oxides are leached out according to the invention. The glass composition of this bioactive carrier, as well as the bioactive carrier, for those described below

Settlement experiments were used, or the chemical components of this glass are (in mass%):

- Si0 ₂ 62.7

- P2O5 7.0

B2O3 2.0

- Na ₂ 0 25.6

- K ₂ 0 1.0

- Ti0 ₂ 0.12

- Zr0 ₂ 0.1

- CuO 0.02

- ZnO 0.06

- NaCI 0.3

- NaF 0.2

Na ₂ S0 ₄ 0.4 - KNOs 0.5

To produce this bioactive carrier, a raw material mixture of highly pure, powdery and mainly oxidic raw materials corresponding to the selected composition, for example the above composition, is first weighed out in one, as shown in FIG. 3

Homogenized ceramic mortar and then to avoid

Melt contamination melted in a platinum-gold crucible (see steps a and b in Fig. 3). After melting at the melting temperature of approx. 1300 ° C. and a holding time of approx. 6 hours, the relevant parameters depending on the glass composition selected, the melt is preferably poured into a preheated steel mold in order to

To form vitreous. After casting and demoulding, the vitreous is transferred to another furnace to relieve any intrinsic stress.

Then (see step c in FIG. 3) a glass powder is produced from the glass body, for which purpose the glass body is broken and comminuted in a mill, preferably a zirconium oxide ball mill. After grinding, it will

Glass powder sieved and fractionated accordingly, preferably only very fine glass powder with a grain size up to approx. 40 pm being used for the further production of the bioactive carrier or the scaffold.

In step d, a slip, ie a suspension of the or

containing the fractionated glass powder. This is done with the addition of one or more dispersants and at least one binder, such as polyvinyl alcohol in an aqueous or organic

Solvents such as isopropanol. The slip is homogenized with a stirring device such as a magnetic stirrer fish. The slip produced in this way is then applied (see step e in FIG. 3) to a porous sponge carrier made of a polymer such that the slip or the suspension covers the surface of the sponge carrier, hence the glass powder dispersed in the slip

Sponge carrier surface covered.

In a subsequent heat treatment step (see step f according to FIG. 3), the covered sponge carrier is heated. The temperature is increased until a sintering temperature for the glass powder is reached, which is in the range between 600 - 740 ° C. At this temperature, the polymer sponge carrier, preferably consisting of polyurethane, degrades, on the one hand, that is, the organic components are oxidized and converted into the gaseous phase. At the same time, the powder particles sinter together without crystallizing. This is done by viscous flow in the interface area. As a result of this sintering process, with simultaneous degradation of the sponge carrier, the glass particles sintered to one another depict the sponge structure in a proportionally reduced manner, that is to say that the resulting sintered glass body is consequently an exact, albeit proportionally reduced, image of the porous sponge carrier originally used, and is therefore also correspondingly porous. The result of this is that the three-dimensional vitreous body or bioactive carrier has a corresponding three-dimensional bar structure with large pores located between the bars.

The bioactive carrier shown in Fig. 1 is not leached, that is, it is the result of the process after the end of the heat treatment.

As can be seen, the bioactive carrier 1 shown in FIG. 1 has a three-dimensional web framework 2 with a large number of individual webs 3, which form a porous but stable framework which has a corresponding number of pores or passages 4 defined via the webs. The webs and the pores or passages have, depending on the sponge carrier used, which, as described, forms the basis for the vitreous body depicting it,

corresponding thicknesses or diameters. A typical web diameter is in the range from 20 to 80 mm, a pore has a size in the range from 100 to 500 μm. Such a bioactive carrier 1 or this web scaffold 2 serves, as will be discussed below, as the basis for the colonization with living cells. As described, FIG. 1 shows the bioactive carrier 1 before leaching.

According to the invention, after the manufacture of this glass bar framework 2, the bioactive carrier 1 is then leached out in a next step g (see FIG. 3) in order to remove alkaline earth metal oxides, i.e. the sodium contained in the glass, at least in the area of the bars 3, preferably over the entire bar cross section - and leach potassium ions. For this purpose, the bioactive carrier 1 according to FIG. 1 is placed, for example, in 1 normal salt column at, for example, 37 ° C. There is an ion exchange between the alkali ions Na ⁺ and K ⁺ and the H ⁺ contained in the acid. In addition, H2O is embedded in the glass structure, so that depending on how long the acid treatment lasts and how deep it takes

Leaching takes place, resulting in a correspondingly water-leached structure, which is however stable and firm. With web thicknesses in the range of less than 10 pm, a complete ion exchange of the alkali ions of a web can be achieved in approx. 8 - 12 days.

After the leaching has ended, a bioactive carrier 1 is rinsed and then in a buffer solution, for example a TRIS buffer,

kept until it is settled.

FIG. 2 shows a microscopically enlarged image of a leached bioactive carrier 1 or the bar framework 2. The leaching out of the alkali ions and the incorporation of the water into the bar framework change its glass structure or composition, which also changes optically in the form of the slightly milky bars 3 shows. However, the web structure as such is retained, as is the pore structure, as the comparison between FIGS. 1 and 2 clearly shows.

As described, the bioactive carrier 1 according to the invention is reduced by leaching at least at the edge in its content of alkali ions or alkali oxides. 4 and 5 show an element scan in one

Scanning electron microscope using EDAX over an edge region of a broken web of a bioactive carrier leached according to the invention. 4 shows a microscopically enlarged view of a partial area of a web 3, a scan line S is shown, along which the element scan was carried out from a central area of the web 3 to its edge 5. The result of the element scan is shown in FIG. 5, only the values determined for the silicon and sodium ions being plotted here.

In the upper diagram area in FIG. 5, the path of the is along the abscissa

Line scans plotted in micrometers, along the ordinate the proportion of Si and Na ions in the form of the count rate of the EDAX measurement. Below the diagram is a section of the image according to FIG. 4 in which the scan is running

Position reference shown.

The upper, solid line shows the Si concentration, the lower, dashed line the Na concentration. In the web volume, starting from a scan path of 0 pm to a scan path of approx. 22 pm, the concentrations remain in the

Essentially constant, apart from the usual local fluctuations. From a scan path of approx. 22 pm, however, a clear drop in the Na concentration can be seen, the value of approx. 25 drops rapidly to a value of significantly below 10. The Na concentration decreases in the course of the further

Element scans between points 1 and 2, which are in the under the

Line diagram shown section of the microscope image of Fig. 4 are drawn along the scan line even further. Point 2 marks the edge of the web. It can be seen that there is an extreme reduction in the Na ion concentration in the area near the edge between positions 1 and 2.

If the leaching had lasted for a longer period of time, the leaching front would move further into the web volume, as described an almost complete leaching over the entire web cross-section is possible. On

Degree of leaching, as shown by way of example in FIGS. 4 and 5, can be achieved after a leaching time of approximately 12 hours.

The potassium ion content, even if not shown in FIG. 5, would be similar, here too there would be a significant drop in the range in which the acid is diffused, although the potassium content is generally significantly lower than the sodium content.

As described, a bioactive carrier according to the invention with a leached out surface layer or a deeply leached out bridge structure serves to colonize and multiply cells living on it. To demonstrate the particular suitability of the bioactive carrier according to the invention, colonization experiments were carried out with three different cell types, namely with porcine

Gel cchondrocytes, with human mesenchymal stromal cells that can be prospectively differentiated into corpus cells, and with human skin fibroblasts. 6 - 11 show two microscopic images for each cell type at different times, which clearly show the colonization and growth process with the different cell types.

6 and 7 show two images of a bioactive carrier populated with porcine articular chondrocytes. FIG. 6 shows the state of colonization 24 hours after the application of the porcine articular chondrocytes, FIG. 7 shows the state of colonization after 21 days.

8 and 9 show two images of the settlement of one

bioactive carrier according to the invention with human mesenchymal

Stromal cells, FIG. 8 showing the condition 24 hours after the application of the stromal cells and FIG. 9 the condition after 7 days of colonization.

Correspondingly, FIGS. 10 and 11 show the colonization of a bioactive carrier according to the invention with human skin fibroblasts, once after 24 hours of colonization (FIG. 10), once after 7 days of colonization (FIG. 11).

Appropriate preparations were made to carry out the tests

Cell suspensions are pipetted onto appropriately prepared bioactive carriers of the glass composition mentioned at the beginning and then incubated in an incubator. The tests were carried out as follows: Sterilize the bioactive carrier stored in TRIS-buffered saline (TBS) in an autoclave at 121 ° C for 20 minutes in a covered

Glass container after the TBS solution has been removed

- After cooling, rinsing the bioactive carrier with phosphate-buffered

Saline solution (PBS, without calcium and magnesium ions), then aspirating the liquid from the bioactive carriers through a sterile non-woven fabric so that the carriers are as dry as possible for the settlement

Place the bioactive carrier in a sterile bench in a sterile 50 ml

Bioreactor tube with filter cap

Treatment of cells intended for scaffold colonization

(Joint chondrocytes, mesenchymal stromal cells and fluman fibroblasts) after a rinsing step with PBS for 5 minutes in an incubator with

Trypsin / EDTA solution (0.05 / 0.02%) to detach the cells from the culture medium of the cell culture flasks

Articular chondrocytes and human fibroblasts: addition of 5 ml of culture medium containing Fletal's F-12 / Dulbecco's Modified Eagle's (DME) medium containing 50% fetal calf serum (FCS) [DMEM] medium 1: 1), 50 IU / ml streptomycin, 50 IU / ml penicillin, 2.5 pg / ml amphotericin B, non-essential amino acids and 25 pg / ml ascorbic acid

Mesenchymal stromal cells: addition of 5 ml stem cell-specific medium (FG0445 / Dulbecco’s Modified Eagle’s [DMEM] / FIG) with 5%

Platelet lysate (PL), 50 IU / ml streptomycin, 50 IU / ml penicilin, 2.5 pg / ml amphotericin B, 100 mM sodium pyruvate and 5000 U / ml heparin

- Stop the detachment process by adding 5 ml of 10% FCS

Medium, the FCS terminating the enzymatic activity of the trypsin. Centrifugation of the cell suspension at 300 rpm or 484 g for 5 minutes of resuspension of the cell pellet resulting from the centrifugation in 1 ml of culture medium

- Mix 10 pl of trypan blue solution (1%) with 10 pl of cell suspension so that dead cells stain

Pipette 10 pl from this mixture into a Neubauer cell chamber and optically count the living cells Pipette the cells in a concentration of 50,000 to 1,000,000 cells in a small volume (0.5 - 1 ml) onto a bioactive carrier, which is positioned in the conically tapered area of the bioreactor tube

Static incubation for two hours at 37 ° C and 5% CO2 in the standing bioreactor tube in the incubator to enable the respective cells to first adhere

Fill the culture medium to a volume of 5 ml to ensure a supply in the subsequent dynamic culture

Cultivation of the populated bioactive carriers in the incubator at 37 ° C and 5% CO2 on an orbital shaker (dynamic)

if the incubation period is longer, change the culture medium every three days

As indicated in FIGS. 6-11, incubation times of different lengths were carried out depending on the cell type. One bioactive carrier each with one cell type was colonized for 24 hours, one carrier gelled with chondrocytes was colonized for 21 days, while the two bioactive carriers colonized with the main fibroblasts and the stromal cells were colonized for 7 days. The latter is due to the fact that these cells grow significantly faster than gels of chchrocytes.

In order to visualize the living cells, the colonization of which is shown in FIGS. 6-11, in a laser scanning microscope, it is necessary to carry out vitality staining. This was done as follows:

Preparation of a vitality staining solution from 5 pl fluorescein diacetate (FDA) solution, 1 pl propidium iodide (PI) solution and 1000 ml PBS

Removal of the respective bioactive carrier from the dynamic

Cultivation in the incubator

Pipette 50 ml vitality solution onto the populated bioactive carrier. Examination of the cells: due to the staining with the vitality solution, living cells can be differentiated green and dead cells red on the bioactive carrier using a confocal laser scanning microscope The PBS serves as a buffer and FDA is used as the dye. This dye penetrates the living cells and is enzymatically converted into a fluorescent compound that is excited with a wavelength of 525 nm. PI intercalates into the DNA of dead cells because their cell and nuclear membranes become permeable. PI is excited with a wavelength of 600 nm.

6-11 are the living cells of the respective cell types

(Joint chondrocytes, flaut fibroblasts, stromal cells). This means that the light areas are the areas of the bioactive carrier that are populated with the living, multiplying cells. The dark areas are primarily non-populated areas of the wearer.

6 and 7 show laser scanning microscope images of a bioactive carrier with porcine articular chondrocytes. As FIG. 6 shows, the carrier was already populated with living cells over a large area within 24 hours after the cell solution had been pipetted on. In the following 20 days, a continuous cell growth or a continuous cell multiplication began, as FIG. 7 clearly shows. The bioactive carrier is significantly more extensive or more densely populated, the uninhabited areas, or the pores or zones between the webs, are significantly smaller, sometimes completely filled.

This means that the bioactive carrier according to the invention demonstrably enables active cell growth or active cell multiplication of this cell type.

As described in the introduction, there is the problem with previously known and used bioactive carriers due to the formation of flydroxyapatite that, in particular with articular chondrocytes, they can change the cell type due to the presence of flydroxylapatite, ie differentiate in the direction of a bone cell. This is not the case with the leached bioactive carrier according to the invention, which does not have such a hydroxyapatite layer, which means that the articular chondrocytes do not change their cell type during growth. To check this, a joint was used for the joint chondrocytes

Immunolabeling of type II collagen, a cartilage cell identification marker. In addition, a non-specific collagen type was marked for comparison (type I collagen). This was done as follows:

Removal of a bioactive carrier populated with articular chondrocytes from the culture in the incubator

Fixation in 4% cold paraformaldehyde (PFA)

Rinsing the carrier with 1x Tris-buffered saline (TBS)

- 20 minutes incubation in a blocking solution (TBS 0.1% TritonX-100, 5%

Donkey serum)

Dilute primary antibodies (AK) with blocking solution

Pipette 50 pl of the primary AK solution onto the articular chondrocyte support and incubate at 4 ° C for 12 hours

- rinsing of the carrier after incubation with TBS (1 s)

Incubation of the carrier in the dark with secondary AK (donkey anti-rabbit IgG), coupled with the fluorescent marker Alexa Fluor 555 for type II collagen and Alexa Fluor 488 coupled donkey anti-goat IgG for type I collagen for one hour

- Staining of the cell nuclei via one contained in the blocking solution

Fluorescent dye DAPI (stock solution: 20 mg / ml) in the dilution 1: 100 after incubation for 60 minutes at room temperature in the dark, rinsing the support 3 times for 5 minutes with TBS (1x)

Assessment of immunohistochemical staining with a DMi8 confocal laser scanning microscope

With the DMi8 confocal laser scanning microscope, it is possible to make fluorescent-labeled markers visible. The PFA solution (paraformaldehyde) is used to fix the cells on the support. The blocking solution serves to prevent non-specific binding of the antibodies to be detected and to achieve permeabilization of the cell membrane.

TBS (TRIS-buffered saline) is used to rinse off unbound

Antibodies or reagents.

The antibody solution (AK) is used to detect the desired protein, here collagen type II and type I.

DAPI (4 ', 6-diamidino-2-phenylindole) is used for core staining.

Type II collagen is only contained in gels of kchondrocytes. Consequently, if the cells show collagen type II after the prolonged colonization, i.e. if they have the corresponding positive immune labeling, this proves that they are articular chondrocytes that were originally pipetted on and have therefore multiplied.

Appropriate investigations of the immune labeling with the laser scanning microscope have shown that the cells have type II collagen and consequently the articular chondrocytes do not change their cell type. That means the

leached bioactive carrier according to the invention has no influence on the cell type, unlike previously known bio glasses, in which the

Hydroxyapatite layer forms. The bioactive carrier according to the invention is consequently completely inert with regard to the cell types to be grown.

8 and 9 show two microscope images of bioactive carriers after 24 hours and 7 days of colonization with mesenchymal stromal cells. Here, too, it shows on the basis of FIG. 8 that basic settlement has already taken place at the beginning of the incubation period, but the settled areas are not yet too large. In contrast, FIG. 9 shows that the cells have proliferated rapidly. It can be seen that the bioactive carrier is largely populated after only 7 days of incubation. Accordingly, this cell type also grows actively and at a high rate of increase on the leached bioactive carrier according to the invention.

This is also shown in FIGS. 10 and 11, which show a bioactive carrier with flaut fibroblasts after 24 hours and 7 days of colonization. Already after 24 hours, a relatively large-scale settlement appears, the increase is steadily increasing. As shown in FIG. 11, the carrier is almost completely populated after 7 days.

This experiment also shows that the leached bioactive carrier according to the invention enables active and rapid growth of dead fibroblasts.

In addition, the experiments show that the bioactive carrier according to the invention enables the growth of different cell types. This means that it is not selectively only suitable for a certain cell type.

Below is a list of those carried out as part of the

Settlement experiments and the materials and equipment used for immunolabelling are shown:

Claims

claims

1. Bioactive carrier for the settlement of living cells, with a porous three-dimensional glass framework that contains

Alkaline earth metal oxides of a maximum of 2.0 mass% and that at least superficially by leaching in its content

Alkali metal oxides is reduced.

2. Bioactive carrier according to claim 1, characterized in that the bridge structure is reduced by alkali oxide over the bridge cross section.

3. Bioactive carrier according to claim 1 or 2, characterized in that the content of alkali metal oxides superficially or over the

Web cross-section is a maximum of 5 mass%.

4. Bioactive carrier according to one of the preceding claims, characterized in that the pore size of the web frame is between 20-300 miti, in particular between 50-200 pm.

5. Bioactive carrier according to one of the preceding claims, characterized in that the thickness of the webs is between 30-120 pm, in particular 50-100 pm.

6. Bioactive carrier according to one of the preceding claims, characterized in that it contains (in mass%):

- Si0 ₂ 77.5 - 85.0

- P2O5 8.5 - 12.0

B2O3 0.5-3.4

Na ₂ 0 + K2O <6.5

7. Bioactive carrier according to claim 6, characterized in that it contains (in mass%):

- Si0 ₂ 79.5 - 83.0 P2O5 9.0 - 11.7

B2O3 0.65 - 3.25

Na2Ü + K2O <6.5 8. Bioactive carrier according to claim 6 or 7, characterized in that it additionally contains (in mass%):

- T1O2 0.1 - 0.5 and / or

Zr0 ₂ 0.1 - 0.4

Bioactive carrier according to one of claims 6 to 8, characterized

marked that it additionally contains (in mass%):

- CuO maximum 0.04 and / or

- ZnO maximum 0.1 and / or

- NaCI maximum 0.5 and / or

- NaF maximum 0.4 and / or

- Na2S0 ₄ maximum 0.6 and / or

- KNO3 maximum 0.6

10. Glass composition for the production of a bioglass, in particular a bioactive carrier according to one of claims 1 to 9, containing (in

Dimensions%):

- S1O2 60.0 - 65.5

- P2O5 6.7 - 9.3

B2O3 0.4-2.6

- Na ₂ 0 25.5 - 28.5

- K ₂ 0 0.45 - 1.55

and an alkaline earth metal oxide content of <2.0% by mass.

11. Glass composition according to claim 10, containing (in mass%):

- Si0 ₂ 61, 5 - 64.0

- P2O5 7.0 - 9.0

- B2O3 0.5 - 2.5

- Na ₂ 0 26.0 - 28.0 K ₂ 0 0.5 - 1.5

and an alkaline earth metal oxide content of <2.0% by mass.

Glass composition according to claim 11, additionally comprising (in mass%):

- T1O2 0.1 - 0.5 and / or

- Zr0 ₂ 0.1 - 0.4

Glass composition according to one of claims 10 to 12, additionally containing (in mass%):

- CuO maximum 0.04 and / or

- ZnO maximum 0.1

Glass composition according to one of claims 10 to 13, additionally containing (in mass%):

- NaCI maximum 0.5 and / or

- NaF maximum 0.4

Glass composition according to one of claims 10 to 14, additionally containing (in mass%):

- Na2S0 ₄ maximum 0.6 and / or

- KNO3 maximum 0.6

Glass composition according to one of Claims 10 to 15, containing (in mass%):

- Si0 ₂ 62.7

- P2O5 7.0

B2O3 2.0

- Na ₂ 0 25.6

- K ₂ 0 1.0

- Ti0 ₂ 0.12

- Zr0 ₂ 0.1

- CuO 0.02 - ZnO 0.06

- NaCI 0.3

- NaF 0.2

Na ₂ S0 ₄ 0.4

- KNOs 0.5

A method for producing a bioactive carrier according to one of claims 1 to 9, comprising the following steps:

- Use of a glass composition according to one of claims 10 to 16 and production of a glass melt therefrom

- Production of a glass body from the glass melt

- Grinding the glass body to form a glass powder

- Formation of a suspension from the glass powder and an aqueous or organic solvent

- Applying the suspension to a porous sponge carrier made of a polymer while covering the surface of the sponge carrier with the glass powder

- Heat treatment of the coated sponge carrier for thermal decomposition of the sponge carrier and simultaneous sintering of the glass powder to form a porous three-dimensional framework

- Leaching of the bridge scaffold to reduce the content

Alkali metal oxides in the bridge framework.

A method according to claim 17, characterized in that a glass powder fraction with a grain size <100 miti, in particular <40 pm is formed from the glass powder, with which the suspension is produced.

A method according to claim 17 or 18, characterized in that isopropanol is used as the solvent.

20. The method according to any one of claims 17 to 19, characterized in that the suspension is produced using at least one dispersant and / or at least one binder. 21. The method according to claim 20, characterized in that as

Binder polyvinyl alcohol is used.

22. The method according to any one of claims 17 to 21, characterized in that a sponge made of polyurethane is used as the sponge carrier.

23. The method according to any one of claims 17 to 22, characterized in that the heat treatment is carried out at a temperature of 550-800 ° C, 600-740 ° C. 24. The method according to any one of claims 17 to 23, characterized in that the leaching is carried out with hydrochloric acid, in particular 0.1 molar hydrochloric acid.

25. The method according to claim 23 or 24, characterized in that the leaching takes place at a temperature between 30 - 40 °, in particular at 37 °, for a period of several days.