WO2019243066A1 - Support bioactif - Google Patents

Support bioactif Download PDF

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Publication number
WO2019243066A1
WO2019243066A1 PCT/EP2019/064758 EP2019064758W WO2019243066A1 WO 2019243066 A1 WO2019243066 A1 WO 2019243066A1 EP 2019064758 W EP2019064758 W EP 2019064758W WO 2019243066 A1 WO2019243066 A1 WO 2019243066A1
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WIPO (PCT)
Prior art keywords
glass
mass
maximum
bioactive carrier
carrier
Prior art date
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PCT/EP2019/064758
Other languages
German (de)
English (en)
Inventor
Armin Lenhart
Lukas Forchheimer
Gundula Schulze-Tanzil
Clemens Gögele
Original Assignee
Technische Hochschule Nürnberg Georg Simon Ohm
Klinikum Nürnberg Medical School GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Application filed by Technische Hochschule Nürnberg Georg Simon Ohm, Klinikum Nürnberg Medical School GmbH filed Critical Technische Hochschule Nürnberg Georg Simon Ohm
Priority to EP19729515.7A priority Critical patent/EP3810216A1/fr
Publication of WO2019243066A1 publication Critical patent/WO2019243066A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/02—Inorganic materials
    • A61L27/10—Ceramics or glasses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3817—Cartilage-forming cells, e.g. pre-chondrocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/58—Materials at least partially resorbable by the body
    • C—CHEMISTRY; METALLURGY
    • C03—GLASS; MINERAL OR SLAG WOOL
    • C03B—MANUFACTURE, SHAPING, OR SUPPLEMENTARY PROCESSES
    • C03B19/00—Other methods of shaping glass
    • C03B19/06—Other methods of shaping glass by sintering, e.g. by cold isostatic pressing of powders and subsequent sintering, by hot pressing of powders, by sintering slurries or dispersions not undergoing a liquid phase reaction
    • C—CHEMISTRY; METALLURGY
    • C03—GLASS; MINERAL OR SLAG WOOL
    • C03C—CHEMICAL COMPOSITION OF GLASSES, GLAZES OR VITREOUS ENAMELS; SURFACE TREATMENT OF GLASS; SURFACE TREATMENT OF FIBRES OR FILAMENTS MADE FROM GLASS, MINERALS OR SLAGS; JOINING GLASS TO GLASS OR OTHER MATERIALS
    • C03C21/00—Treatment of glass, not in the form of fibres or filaments, by diffusing ions or metals in the surface
    • C03C21/001—Treatment of glass, not in the form of fibres or filaments, by diffusing ions or metals in the surface in liquid phase, e.g. molten salts, solutions
    • C03C21/006—Treatment of glass, not in the form of fibres or filaments, by diffusing ions or metals in the surface in liquid phase, e.g. molten salts, solutions to perform an exchange of the type Xn+ ----> nH+
    • C—CHEMISTRY; METALLURGY
    • C03—GLASS; MINERAL OR SLAG WOOL
    • C03C—CHEMICAL COMPOSITION OF GLASSES, GLAZES OR VITREOUS ENAMELS; SURFACE TREATMENT OF GLASS; SURFACE TREATMENT OF FIBRES OR FILAMENTS MADE FROM GLASS, MINERALS OR SLAGS; JOINING GLASS TO GLASS OR OTHER MATERIALS
    • C03C3/00—Glass compositions
    • C03C3/04—Glass compositions containing silica
    • C03C3/076—Glass compositions containing silica with 40% to 90% silica, by weight
    • C03C3/11—Glass compositions containing silica with 40% to 90% silica, by weight containing halogen or nitrogen
    • C03C3/112—Glass compositions containing silica with 40% to 90% silica, by weight containing halogen or nitrogen containing fluorine
    • C—CHEMISTRY; METALLURGY
    • C03—GLASS; MINERAL OR SLAG WOOL
    • C03C—CHEMICAL COMPOSITION OF GLASSES, GLAZES OR VITREOUS ENAMELS; SURFACE TREATMENT OF GLASS; SURFACE TREATMENT OF FIBRES OR FILAMENTS MADE FROM GLASS, MINERALS OR SLAGS; JOINING GLASS TO GLASS OR OTHER MATERIALS
    • C03C4/00—Compositions for glass with special properties
    • C03C4/0007—Compositions for glass with special properties for biologically-compatible glass
    • C—CHEMISTRY; METALLURGY
    • C03—GLASS; MINERAL OR SLAG WOOL
    • C03C—CHEMICAL COMPOSITION OF GLASSES, GLAZES OR VITREOUS ENAMELS; SURFACE TREATMENT OF GLASS; SURFACE TREATMENT OF FIBRES OR FILAMENTS MADE FROM GLASS, MINERALS OR SLAGS; JOINING GLASS TO GLASS OR OTHER MATERIALS
    • C03C8/00—Enamels; Glazes; Fusion seal compositions being frit compositions having non-frit additions
    • C03C8/14—Glass frit mixtures having non-frit additions, e.g. opacifiers, colorants, mill-additions
    • C03C8/16—Glass frit mixtures having non-frit additions, e.g. opacifiers, colorants, mill-additions with vehicle or suspending agents, e.g. slip
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/08—Methods for forming porous structures using a negative form which is filled and then removed by pyrolysis or dissolution
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/18—Modification of implant surfaces in order to improve biocompatibility, cell growth, fixation of biomolecules, e.g. plasma treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/06—Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus

Definitions

  • 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
  • tissue engineering This process of obtaining tissue by culturing cultivated cells in a suitable biomaterial is called "tissue engineering”.
  • 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.
  • 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.
  • Tissue formation is of major importance:
  • 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.
  • 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.
  • 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).
  • growth stimulators such as growth factors, ions, but also a suitable scaffold topology.
  • ECM extracellular matrix
  • 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.
  • bioactive carriers or bio glasses described there are based on alkaline earth and phosphate
  • 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.
  • 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.
  • 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
  • the glass composition is characterized in that there is no addition of any alkaline earth metal oxide.
  • 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.
  • 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.
  • Glass carrier also dissolves due to the hydrolytic acid-alkali attack of the surrounding medium given an in vivo use.
  • one of the central features of the bioactive carrier or bioglass according to the invention is that a superficial strong one
  • 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.
  • Hydrochloric acid for example at 37 ° C instead.
  • 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.
  • 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 ⁇ m. 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.
  • 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.
  • the bioactive carrier in mass%) contains:
  • 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
  • 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
  • the bioactive carrier can contain (in mass% based on the dry substance):
  • the bioactive carrier can additionally contain (in mass% based on the dry substance): - Ti0 2 0.1 - 0.5 and / or
  • bioactive carrier can additionally contain (in mass% based on the dry substance):
  • the invention further relates to a specific glass composition according to the invention which is suitable for the
  • Glass composition contains (in mass%):
  • 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
  • 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.
  • the glass composition can contain (in mass%):
  • alkaline earth metal oxides 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.
  • the glass composition according to the invention can additionally contain (in mass%): T1O2 0.1-0.5 and / or
  • 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.
  • the glass composition additionally contains according to the invention (in mass%):
  • 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
  • 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.
  • the glass composition according to the invention additionally contains (in mass%): - Na 2 S0 4 max. 0.6 and / or
  • These two compounds are preferably added to the batch in order to homogenize and refine the melt.
  • 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%):
  • the invention further relates to a method for producing a bioactive
  • Vitreous is poured.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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 ⁇ m, 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.
  • 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 Kunststoff GmbH under the
  • 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
  • Fig. 2 shows the bioactive carrier from Fig. 1 after the leaching of
  • Fig. 3 is a diagram for explaining the essential steps of the
  • Fig. 5 is a diagram of a line scan over the fracture surface of the
  • FIG. 6 shows a microscopic image of living porcine articular chondrocytes on a bioactive carrier according to the invention after 24 hours
  • 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
  • FIG. 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.
  • 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
  • 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
  • vitreous After casting and demoulding, the vitreous is transferred to another furnace to relieve any intrinsic stress.
  • 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.
  • step d a slip, ie a suspension of the or
  • fractionated glass powder 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 solvent, such as polyvinyl alcohol in an aqueous or organic solvent, such as polyvinyl alcohol in an aqueous or organic solvent, such as polyvinyl alcohol in an aqueous or organic solvent, such as polyvinyl alcohol in an aqueous or organic solvent
  • 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
  • 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.
  • 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.
  • the powder particles sinter together without crystallizing. This is done by viscous flow in the interface area.
  • 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 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.
  • 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,
  • FIG. 1 shows the bioactive carrier 1 before leaching.
  • 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.
  • the bioactive carrier 1 according to FIG. 1 is placed, for example, in 1 normal salt column at, for example, 37 ° C.
  • H2O is embedded in the glass structure, so that depending on how long the acid treatment lasts and how deep it takes
  • a bioactive carrier 1 is rinsed and then in a buffer solution, for example a TRIS buffer,
  • 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.
  • the web structure as such is retained, as is the pore structure, as the comparison between FIGS. 1 and 2 clearly shows.
  • 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
  • the upper, solid line shows the Si concentration
  • the lower, dashed line the Na concentration
  • 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.
  • 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.
  • 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.
  • FIG. 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.
  • bioactive carrier according to the invention with human mesenchymal
  • FIG. 8 showing the condition 24 hours after the application of the stromal cells
  • FIG. 9 the condition after 7 days of colonization.
  • 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).
  • 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
  • DME Dulbecco's Modified Eagle's
  • FCS fetal calf serum
  • Mesenchymal stromal cells addition of 5 ml stem cell-specific medium (FG0445 / Dulbecco’s Modified Eagle’s [DMEM] / FIG) with 5%
  • 6-11 are the living cells of the respective cell types
  • FIG. 6 and 7 show laser scanning microscope images of a bioactive carrier with porcine articular chondrocytes.
  • the carrier was already populated with living cells over a large area within 24 hours after the cell solution had been pipetted on.
  • 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.
  • bioactive carrier according to the invention demonstrably enables active cell growth or active cell multiplication of this cell type.
  • 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)
  • the PFA solution paraformaldehyde
  • the blocking solution serves to prevent non-specific binding of the antibodies to be detected and to achieve permeabilization of the cell membrane.
  • TBS TBS-buffered saline
  • the antibody solution (AK) is used to detect the desired protein, here collagen type II and type I.
  • DAPI 6-diamidino-2-phenylindole
  • 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.
  • leached bioactive carrier according to the invention has no influence on the cell type, unlike previously known bio glasses, in which the
  • the bioactive carrier according to the invention is consequently completely inert with regard to the cell types to be grown.
  • FIG. 8 and 9 show two microscope images of bioactive carriers after 24 hours and 7 days of colonization with mesenchymal stromal cells.
  • FIG. 8 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.
  • 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.
  • FIGS. 10 and 11 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.
  • 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.

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Abstract

L'invention concerne un support bioactif pour l'implantation de cellules vivantes, comprenant une armature de barre poreuse tridimensionnelle en verre, qui présente une teneur en oxydes de métaux alcalino-terreux de 2,0 % en poids au maximum et dont la teneur en oxydes de métaux alcalino-terreux est réduite au moins en surface par lixiviation.
PCT/EP2019/064758 2018-06-21 2019-06-06 Support bioactif WO2019243066A1 (fr)

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DE102018114946.8A DE102018114946B3 (de) 2018-06-21 2018-06-21 Bioaktiver Träger
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Citations (3)

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Publication number Priority date Publication date Assignee Title
WO2011161422A1 (fr) 2010-06-25 2011-12-29 Queen Mary And Westfield College Composition de verre bioactif
WO2014168631A1 (fr) 2013-04-12 2014-10-16 Mosci Corp. Structure de verre bioactif, et son procédé de fabrication
WO2016089731A1 (fr) 2014-12-04 2016-06-09 3M Innovative Properties Company Compositions antimicrobiennes comprenant un verre bio-actif

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Publication number Priority date Publication date Assignee Title
WO2011161422A1 (fr) 2010-06-25 2011-12-29 Queen Mary And Westfield College Composition de verre bioactif
WO2014168631A1 (fr) 2013-04-12 2014-10-16 Mosci Corp. Structure de verre bioactif, et son procédé de fabrication
WO2016089731A1 (fr) 2014-12-04 2016-06-09 3M Innovative Properties Company Compositions antimicrobiennes comprenant un verre bio-actif

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Title
DATABASE MEDLINE [online] US NATIONAL LIBRARY OF MEDICINE (NLM), BETHESDA, MD, US; 2001, KUBOKI Y ET AL: "Geometry of carriers controlling phenotypic expression in BMP-induced osteogenesis and chondrogenesis.", XP002794001, Database accession no. NLM11314788 *
KUBOKI Y ET AL: "Geometry of carriers controlling phenotypic expression in BMP-induced osteogenesis and chondrogenesis.", THE JOURNAL OF BONE AND JOINT SURGERY. AMERICAN VOLUME 2001, vol. 83-A Suppl 1, no. Pt 2, 2001, pages S105 - S115, ISSN: 0021-9355 *

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