WO2015173020A1

WO2015173020A1 - 3d cell printing of bioglass-containing scaffolds by combination with cell-containing morphogenically active alginate/gelatin hydrogels

Info

Publication number: WO2015173020A1
Application number: PCT/EP2015/059333
Authority: WO
Inventors: Werner E.G. Müller; Xiaohong Wang; Heinz C. Schröder
Original assignee: Müller Werner E G; Xiaohong Wang; Schröder Heinz C
Priority date: 2014-05-12
Filing date: 2015-04-29
Publication date: 2015-11-19
Also published as: GB201408402D0

Abstract

The present invention relates toa combined system for three-dimensional (3D) bioprinting of cells, especially bone-forming cells, that comprises(i) a bioprintable and biodegradable cell- containing alginate hydrogelor alginate/gelatin hydrogel,surrounded by (ii) a printable bioglass-containing matrix. The morphogenic activity of the alginate hydrogelor alginate/ gelatin hydrogel, supplemented with the (bio)polymers, polyphosphate-calcium complex or biosilica, is increased in a synergistic way by the bioglass integrated into the inventive bioglass -(bio)polymer -alginate/gelatin hydrogel scaffold, providing this new scaffold with enhanced morphogenetic activity for bone implants.

Description

3D CELL PRINTING OF BIOGLASS-CONTAINING SCAFFOLDS BY

COMBINATION WITH CELL-CONTAINING MORPHOGENICALLY ACTIVE

ALGINATE/GELATIN HYDROGELS

The present invention concerns a combined system for three-dimensional (3D) bioprinting of cells, especially bone-forming cells, that consists of (i) a bioprintable and biodegradable cell- containing alginate hydrogel or alginate/gelatin hydrogel, surrounded by (ii) a printable bioglass-containing matrix. The morphogenic activity of the alginate hydrogel or alginate/ gelatin hydrogel, supplemented with the (bio)polymers, polyphosphate-calcium complex or biosilica, is increased in a synergistic way by the bioglass integrated into the inventive bioglass - (bio)polymer - alginate/gelatin hydrogel scaffold, providing this new scaffold with enhanced morphogenetic activity for bone implants.

Background of the invention

The development of functional, biocompatible biomaterials relies on a thorough understanding of the biology of bone cells and tissue. The knowledge of the composition, development and function of the extracellular matrix and the cells of bone tissue, as well as the growth factors controlling their expression is pivotal for the successful development of bioactive scaffolds (Wang XH, Schroder HC, Wiens M, Ushijima H, Muller WEG (2012) Bio-silica and bio-polyphosphate: applications in biomedicine (bone formation). Curr Opin Biotechnol 23:570-578). Very promising are new developments of fabrication of 3D biocompatible structures that can mimic the properties of the extracellular structure, especially with respect to mechanical support, cellular activity and protein production (reviewed in: Bose S, Vahabzadeh S, Bandyopadhyay A (2013) Bone tissue engineering using 3D printing. Materials Today 16:496-504). Efforts are presently undertaken to bioprint 3D porous bone scaffolds, both by solid free-form fabrication / rapid prototyping (Gibson I, Rosen DW, Stucker B (2010) Additive Manufacturing Technologies - Rapid Prototyping to Direct Digital Manufacturing. Heidelberg: Springer, 462 p) and by bioprinting of 3D tissue units (Tasoglu S, Demirci U (2013) Bioprinting for stem cell research. Trends Biotechnol 31 : 10-19) to create implants that are replaced, with time, by cells from the donors of the implants. Nevertheless, bone cells cannot be embedded into a bioglass scaffold.

3D cell bioprinting

Recently, the inventors have developed a matrix for bone cells, which contains alginate (SchloBmacher U, Schroder HC, Wang XH, Feng Q, Diehl-Seifert B, Neumann S, Trautwein A, Muller WEG (2013) Alginate/silica composite hydrogel as a potential morphogenetically active scaffold for three-dimensional tissue engineering. RSC Adv 3: 11185-11194; Muller WEG, Wang XH, Grebenjuk V, Diehl-Seifert B, Steffen R, SchloBmacher U, Trautwein A, Neumann S, Schroder HC (2013) Silica as a morphogenetically active inorganic polymer: effect on the BMP-2-dependent and RUNX2-independent pathway in osteoblast-like SaOS-2 cells. Biomaterials Sci 1 :669-678).

In this matrix, cells can be readily embedded and bioprinted without losing their proliferation activity. The alginate matrix can be hardened simply by a brief exposure to calcium chloride. In order improve the biological environment for the embedded cells low-melding gelatin can be added (Patent application Great Britain GB 1406840.7. Morphogenetically active hydrogel for bioprinting of bioartificial tissue. Inventors: Miiller WEG, Wang XH, Schroder HC).

In addition, the two natural polymers, either polyphosphate (polyP; comprising various chain length, from 2 to more than 100 phosphate units) or biosilica, can be added to the alginate/gelatin hydrogel.

PolyP increases the potency of SaOS-2 cells and/or mesenchymal stem cells to synthesize mineral deposits and induces the expression of the bone key enzyme, alkaline phosphatase, as well as the cytokine, bone morphogenetic protein 2 (BMP-2), and the major extracellular fibrillar structural molecule, collagen type I (Miiller WEG, Wang XH, Diehl-Seifert B, Kropf K, SchloBmacher U, Lieberwirth I, Glasser G, Wiens M, Schroder HC (2011) Inorganic polymeric phosphate/polyphosphate as an inducer of alkaline phosphatase and a modulator of intracellular Ca²⁺ level in osteoblasts (SaOS-2 cells) in vitro. Acta Biomater 7:2661-2671). In these studies, polyP together with CaCl₂ [polyP^»Ca²⁺-complex] has been used in order to avoid any depletion of Ca²⁺ ions required for mineral formation.

Biosilica is a naturally occurring polymer existing in sponges (reviewed in: Wang XH, Schroder HC, Wiens M, Ushijima H, Miiller WEG (2012) Bio-silica and bio-polyphosphate: applications in biomedicine (bone formation). Curr Opin Biotechnol 23:570-578). Biosilica is formed enzymatically from ortho-silicate by the enzyme silicatein (German Patent No. DE10037270, European Patent No. EP1320624, United States Patent No. US 7,169,589 B2. Silicatein-mediated synthesis of amorphous silicates and siloxanes and their uses. Inventors: Miiller WEG, Lorenz A, Krasko A, Schroder HC) and displays an inductive anabolic bone- forming effect on SaOS-2 cells; e.g. this polymer causes a significant shift of the osteoprotegerin (OPG) : receptor activator of nuclear factor-κΒ ligand (RANKL) ratio (Wiens M, Wang XH, Schroder HC, Kolb U, SchloBmacher U, Ushijima H, Miiller WEG (2010) The role of biosilica in the osteoprotegerin/RANKL ratio in human osteoblast like cells. Bio materials 31 :7716-7725), resulting in an inhibition of the differentiation pathway of pre- osteoclasts into mature osteoclasts. Furthermore, biosilica causes an increased expression of BMP-2 in SaOS-2 cells and shows osteogenic potential (Wiens M, Wang XH, SchloBmacher U, Lieberwirth I, Glasser G, Ushijima H, Schroder HC, Miiller WEG (2010) Osteogenic potential of bio-silica on human osteoblast-like (SaOS-2) cells. Calcif Tissue Int 87:513-524). These results have been supported using human mesenchymal stem cells (Han P, Wu C, Xiao Y (2013) The effect of silicate ions on proliferation, osteogenic differentiation and cell signalling pathways (WNT and SHH) of bone marrow stromal cells. Biomater Sci 1 :379-392).

Bioglass

Bioglasses (bioactive glasses) have been developed as hard, bone-imitating scaffold structures. The present state-of-the-art is reviewed in, Hench LL (2011) Bioactive materials for gene control, in: Hench LL, Jones JR, Fenn MB (eds) New Materials and Technologies for Healthcare. Singapore: World Scientific, pp 25-48; and Jones JR (2013) Review of bioactive glass: from Hench to hybrids. Acta Biomater 9:4457-4486.

Bioglasses turned out to be printable (reviewed in: Luo Y, Wu C, Lode A, Gelinsky M (2013) Hierarchical mesoporous bioactive glass/alginate composite scaffolds fabricated by three- dimensional plotting for bone tissue engineering. Bio fabrication 5:015005, doi: 10.1088/1758-5082/5/1/015005). Several formulations for bioglasses have been proposed, among which 45 S5 Bioglass is the most known one (Hench LL (2006) The story of Bioglass^®. J Mater Sci - Mater Med 17:967- 978); this bioglass:

- has a molar composition of 46.1 mol.% Si0₂, 24.4 mol.% Na₂0, 26.9 mol.% CaO and 2.6 mol.% P₂0₅;

- forms a strong association with bone (Hench LL, Splinter RJ, Allen WC, Greenlee TK (1971) Bonding mechanisms at the interface of ceramic prosthetic materials. J Biomed Mater Res Symp 334: 117-141);

- has osteogenic properties (reviewed in: Jones JR (2013) Review of bioactive glass: from Hench to hybrids. Acta Biomater 9:4457-4486); and

- acts as a platform for the formation of organic-inorganic hybrids, e.g. with poly(methyl methacrylate) (Ravarian R, Wei H, Rawal A, Hook J, Chrzanowski W, Dehghani F (2013) Molecular interactions in coupled PMMA-bioglass hybrid networks. J. Mater Chem Bl : 1835- 1845).

One main problem for a potential application of bioglasses in 3D cell bioplotting is, however, that bone cells cannot be embedded into a bioglass scaffold, especially not in a hard porous sintered solid material.

PolyP

Polyphosphates (polyP) are naturally occurring linear polymers which may consist of two up to several hundreds of phosphate residues (Schroder HC, Miiller WEG, eds. Inorganic Polyphosphates - Biochemistry, Biology, Biotechnology. Prog Mol Subcell Biol 23:45-81).

PolyP can be synthesized both chemically and enzymatically (Kulaev IS, Vagabov V, Kulakovskaya T (2004) The Biochemistry of Inorganic Polyphosphates. New York: John Wiley & Sons Inc).

The enzymatic synthesis of polyP is mediated by polyphosphate kinases (reviewed in: Schroder HC, Lorenz B, Kurz L, Miiller WEG (1999) Inorganic polyP in eukaryotes: enzymes, metabolism and function. In: Inorganic Polyphosphates - Biochemistry, Biology, Biotechnology (Schroder HC, MuUer WEG, eds). Prog Mol Subcell Biol 23:45-81).

The enzymatic degradation of polyP is mediated by exo- and endopolyphosphatases (e.g., Lorenz B, Miiller WEG, Kulaev IS, Schroder HC (1994) Purification and characterization of an exopolyphosphatase activity from Saccharomyces cerevisiae. J Biol Chem 269:22198- 22204).

PolyP is present in bone tissue (Leyhausen G, Lorenz B, Zhu H, Geurtsen W, Bohnensack R, Miiller WEG, Schroder HC (1998) Inorganic polyphosphate in human osteoblast-like cells. J Bone Mineral Res 13:803-812; Schroder HC, Kurz L, MuUer WEG, Lorenz B (2000) Polyphosphate in bone. Biochemistry (Moscow) 65:296-303).

PolyP is a substrate for the principle enzyme involved in bone formation, the bone specific alkaline phosphatase (Lorenz B, Schroder HC (2001) Mammalian intestinal alkaline phosphatase acts as highly active exopolyphosphatase. Biochim Biophys Acta 1547:254-261).

PolyP only becomes bioactive if applied as a complex with Ca²⁺ ions (polyP^»Ca²⁺-complex) (MuUer WEG, Wang XH, Diehl-Seifert B, Kropf K, SchloBmacher U, Lieberwirth I, Glasser G, Wiens M, Schroder HC (2011) Inorganic polymeric phosphate/polyphosphate is an inducer of alkaline phosphatase and a modulator of intracellular Ca²⁺ level in osteoblasts (SaOS-2 cells) in vitro. Acta Biomater 7:2661-2671).

The morphogenetic activity of the polyP^»Ca²⁺-complex can be demonstrated in osteoblast-like SaOS-2 cells (Muller WEG, Wang XH, Diehl-Seifert B, Kropf K, SchloBmacher U, Lieberwirth I, Glasser G, Wiens M, Schroder HC (2011) Inorganic polymeric phosphate/polyphosphate is an inducer of alkaline phosphatase and a modulator of intracellular Ca²⁺ level in osteoblasts (SaOS-2 cells) in vitro. Acta Biomater 7:2661-2671).

The polyP^»Ca²⁺-complex induces not only hydroxyapatite formation but also enhances the expression of the gene encoding BMP-2 in SaOS-2 cells (Wang XH, Schroder HC, Diehl- Seifert B, Kropf K, SchloBmacher U, Wiens M, Muller WEG (2013) Dual effect of inorganic polymeric phosphate/polyphosphate on osteoblasts and osteoclasts in vitro. J Tissue Eng Regen Med, in press, doi: 10.1002/term. l465).

The polyP^»Ca²⁺-complex induces the bone alkaline phosphatase (tissue non-specific alkaline phosphatase), both the activity of this enzyme and the expression of the gene encoding the enzyme.

On the other hand, the polyP^»Ca²⁺-complex inhibits the progression of RAW 264.7 cells into osteoclasts (Wang XH, Schroder HC, Diehl-Seifert B, Kropf K, SchloBmacher U, Wiens M, Muller WEG (2013) Dual effect of inorganic polymeric phosphate/polyphosphate on osteoblasts and osteoclasts in vitro. J Tissue Eng Regen Med, in press, doi: 10.1002/term.l465).

The following patent applications relate to polyP or polyP^»Ca²⁺-complexes; GB 1406840.7. Morphogenetically active hydrogel for bioprinting of bioartificial tissue. Inventors: Muller WEG, Schroder HC, Wang XH; and GB 1403899.6. Synergistic composition comprising quecetin and polyphosphate for treatment of bone disorders. Inventors: Muller WEG, Schroder HC, Wang XH.

Biosilica

The following patents or patent applications are relevant with respect to biosilica:

EP1320624; US7169589B2; DE10037270; CN01813484.X; NZ523474; AU2001289713. Silicatein-mediated synthesis of amorphous silicates and siloxanes and their uses. Inventors: Muller WEG, Lorenz A, Krasko A, Schroder HC.

US6670438B1; Methods, compositions, and biomimetic catalysts for in vitro synthesis of silica, polysilsequioxane, polysiloxane, and polymetallo-oxanes. Inventors: Morse DE, Stucky GD, Deming, TD, Cha J, Shimizu K, Zhou Y.

DE10246186; In vitro and in vivo degradation or synthesis of silicon dioxide and silicones, useful e.g. for treating silicosis or to prepare prosthetic materials, using a new silicase enzyme. Inventors: Muller WEG, Krasko A, Schroder HC.

EP 1546319; Abbau und Modifizierung von Silicaten und Siliconen durch Silicase und Verwendung des reversiblen Enzyms. Inventors: Muller WEG, Krasko A, Schroder HC.

EP1740707; US11579019; DE10352433.9; CA2565118; JP2007509991; Enzym- und Template-gesteuerte Synthese von Silica aus nicht-organischen Siliciumverbindungen sowie Aminosilanen und Silazanen und Verwendung. Inventors: Schwertner H, Mviller WEG, Schroder HC.

EP2248824; Use of silintaphin for the structure-directed fabrication of (nano)composite materials in medicine and (nano)technology. Inventors: Wiens M, Mviller WEG, Schroder HC, Wang X.

DE102004021229.5; EP1740706A1 ; US11/579,020; JP2007509992; CA2565121. Enzymatic method for producing bioactive, osteob last-stimulating surfaces and use thereof. Inventors: MuUer WEG, Schwertner H, Schroder HC.

US60839601; EP2064329. Biosilica-adhesive protein nano-composite materials: synthesis and application in dentistry. Inventors: Mviller WEG, Schroder HC, Geurtsen WK.

WO2010036344; Compositions, oral care products and methods of making and using the same. Inventors: Miller J, Hofer H, Geurtsen W, Lvicker P, Wiens M, Schroder HC, Mviller WEG.

GB1405994.3. Osteogenic material to be used for treatment of bone defects. Inventors: Mviller WEG, Schroder HC, Wang XH.

The present invention relates to a new approach for preparing bioprintable cell-containing scaffolds. The inventors investigated the effect of bioglass (bioactive glass) which is not suitable for embedding bone cells, on growth and mineralization of bone-related SaOS-2 cells encapsulated into a printable and biodegradable alginate/gelatine hydrogel hardened with calcium chloride. In addition, the hydrogel was supplemented either with polyP, given as polyP^»Ca²⁺-complex, or biosilica, enzymatically prepared from ortho-silicate by silicatein, two natural polymers that cause an enhanced mineralization and expression of morphogenetic cytokines by SaOS-2 cells. These hydrogel preparations, together with the SaOS-2 cells, were bioprinted to computer-designed scaffolds. The results revealed that bioglass (nano)particles, if added to the bioprinted scaffolds consisting of the cell containing alginate/gelatin hydrogel, supplemented with either polyP^»Ca²⁺-complex, silica, biosilica, or polyP^»Ca²⁺-complex together with biosilica, did not significantly change the growth of the embedded cells. However, the inventors unexpectedly found that addition of bioglass to the hydrogel significantly enhanced - in a synergistic way - the increase in mineralization caused by these additives, compared to the assays without bioglass, for example, from 2.1- to 3.9-fold (hydrogel with 100 μΜ polyP^»Ca²⁺-complex), from 1.8- to 2.9-fold (hydrogel with 50 μΜ silica), from 2.7- to 4.8-fold (hydrogel with 50 μΜ biosilica) and from 4.1- to 6.8-fold (hydrogel with 100 μΜ polyP^»Ca²⁺-complex and 50 μΜ biosilica). Element analysis by EDS spectrometry of the mineral nodules formed by SaOS-2 revealed an accumulation of O, P, Ca and C, indicating that besides Ca-phosphate, the mineral deposits contain Ca-carbonate.

The results underlying this invention show that bioglass added to an alginate hydrogel or an alginate/gelatin hydrogel or an alginate hydrogel containing another collagen-derived product increases the mineralization of bioprinted SaOS-2 cells.

The inventive, newly developed scaffolds consisting of a bioprintable, solid and cell- compatible inner matrix surrounded by a printable hard and flexible outer matrix containing bioglass provide a suitable strategy for the fabrication of morphogenetically active and biodegradable implants. Detailed description of the invention

Bioglass is a well-established hard, porous basis material for the formation of bone-replacing scaffolds. This material has proven its beneficial role in bone implants and can be printed (Luo Y, Wu C, Lode A, Gelinsky M (2013) Hierarchical mesoporous bioactive glass/alginate composite scaffolds fabricated by three-dimensional plotting for bone tissue engineering. Biofabrication 5:015005, doi: 10.1088/1758-5082/5/1/015005).

In a previous patent application (GB 1406840.7; Morphogenetically active hydrogel for bioprinting of bioartificial tissue. Inventors: Miiller WEG, Wang XH, Schroder HC) the inventors have shown that SaOS-2 cells survive in the alginate matrix after printing but they lose their proliferating capacity. Furthermore, the addition of gelatin to the alginate hydrogel used for embedding the cells provides this matrix with a significant higher stability. Also, the addition of polyP^»Ca²⁺-complex to the alginate/gelatin hydrogel results in cell proliferation.

Then the addition of polyP^»Ca²⁺-complex to the alginate/gelatin hydrogel additionally stabilizes the matrix which becomes substantially harder, the hardness of the SaOS-2 cell- containing and polyP^»Ca²⁺-complex-containing hydrogels decreases with time, whereas in the absence of cells the hardness of the matrix remains high, indicating a metabolic degradation of the polyP^»Ca²⁺-complex, the addition of polyP^»Ca²⁺-complex to the alginate/gelatin hydrogel results in an enhanced mineralization of SaOS-2 cells in the presence of an osteogenic cocktail.

Like polyP^»Ca²⁺-complex, the addition of polymeric silicic acid (silica), another morphogenetically active polymer, or one of its salts to the alginate/gelatin hydrogel increases the mineralization of bone- forming cells, for example SaOS-2 cells, in the presence of an osteogenic cocktail. In addition, combinations of the morphogenetically active polymers, inorganic polyP or a complex of inorganic polyP and divalent metal ions with monomeric silicic acid (orthosilicic acid) or polymeric silicic acid (silica) or its salts can be applied to enhance the mineralization activity of the bone- forming cells.

Now, the inventors have surprisingly found that bioglass (bioactive glass), a widely used material for the preparation of bone implants that is, however, not applicable for embedding of cells, such as bone-forming cells, and not applicable for 3D cell bioprinting,

i) can be used for the bioprinting of cell-containing scaffolds if combined with cell- compatible alginate/gelatine hydrogel, and

ii) is able to increase the morphogenic activity of polyP and biosilica/silica, used as supplements of the alginate/gelatine hydrogel, in a synergistic manner.

In addition, the inventors show that the bone-related SaOS-2 cells can be embedded into an alginate/gelatin matrix which, after supplementation with silica, but especially with polyP and biosilica, allows the cells to proliferate.

Further, the inventors demonstrate that both polyP and biosilica display the potency to enhance the induction of morphogenetically active cytokines, e.g. BMP-2, even if the cells are embedded and bioprinted.

The inventors demonstrate that bioglass added to bioprinted SaOS-2 cells from the outside of the alginate/gelatin hydrogel influences the function of the cells by increasing their proliferation and biomineralization properties. Unexpectedly, this combined effect of polyP and biosilica/silica (component 1) and bioglass (component 2) was synergistic, even if both components are present in two different phases/compartments (3D-printed alginate/gelatin hydrogel phase/compartment and outer phase/compartment). These two phases either can be 3D-printed in parallel or the bioglass matrix can be added from the outside after 3D-printing of the alginate/gelatine hydrogel phase/compartment.

Also the mixing of this bioglass (nano)particles suspension with the cell-containing hydrogel before 3D-bioprinting of the scaffold is suitable to achieve this synergistic effect.

The inventors show that the organic hydrogel scaffold obtained by 3D cell bioprinting is composed of cylinders which leave room not only for the outgrowth of the cells but also for additional scaffold materials, such as a bioglass scaffold, that can likewise be printed.

The inventive material consists of a cell-free, solid and concurrently flexible and integrable printable hard bioglass scaffold which surrounds a more solid and cell compatible inner alginate/gelatin matrix that contains the cells and is likewise bioprintable.

The technology according to this invention can be applied for the fabrication of bioreplacable implants.

The bioglass (bioactive glass) (nano)particles used can be composed of Si0₂:CaO:P₂0₅ or Si0₂:Na₂0:CaO:P₂0₅ of various molar ratios, for example Si0₂:CaO:P₂0₅ of a molar ratio (mol.%) of 55:40:5 or Si0₂:Na₂0:CaO:P₂0₅ of a molar ratio (mol.%) of 46.1 :24.4:26.9:2.6 (45S5 Bioglass^®).

The chain lengths of the polyP molecules added to the alginate/gelatine matrix can be in the range 2 to up to 1000 phosphate units. PolyP molecules consisting of only 2 phosphate units are termed pyrophosphate, polyP molecules with 3 to 10 phosphate units are termed oligophosphates (oligoP). Optimal results were achieved with polyP molecules with an average chain length of approximately 40 phosphate units.

The complex of polyP with divalent cations, preferably Ca²⁺ ions (polyP^»Ca²⁺-complex) is preferentially used to avoid a depletion of divalent cations, in particular calcium ions, by complex formation with polyP, which may interfere with the results.

The preparation of the polyP^»Ca²⁺-complex is state-of-the-art and has previously been described by the inventors (e.g., Miiller WEG, Wang XH, Diehl-Seifert B, Kropf K, SchloBmacher U, Lieberwirth I, Glasser G, Wiens M, Schroder HC. Inorganic polymeric phosphate/polyphosphate as an inducer of alkaline phosphatase and a modulator of intracellular Ca²⁺ level in osteoblasts (SaOS-2 cells) in vitro. Acta Biomater 7:2661-2671, 2011; or patent application EP2489346 Food supplement and injectable material for prophylaxis and therapy of osteoporosis and other bone disease. Inventors: Miiller WEG, Wang X, Schroder HC).

The polymeric silicic acid can be formed by an enzyme or protein involved in bio silica (amorphous, hydrated silicon oxide) metabolism, such as silicatein or a silicatein fusion protein. The silicatein polypeptide or a silicatein fusion protein can be produced using a prokaryotic or eukaryotic expression system, or can be produced synthetically.

The silicatein or silicatein fusion protein can be present together with a suitable substrate (silica precursor) such as water glass, orthosilicic acid, orthosilicates, monoalkoxysilanetriols, dialkoxysilanediols, trialkoxysilanols, tetraalkoxysilanes, alkyl-silanetriols, alkyl-silanediols, alkyl-monoalkoxysilanediols, alkyl-monoalkoxysilanols, alkyl-dialkoxysilanols, or alkyl- trialkoxysilanes.

An additional aspect of the invention concerns a material obtained by one of the methods described above.

The invention will now be described further in the following preferred examples, nevertheless, without being limited thereto. For the purposes of the present invention, all references as cited herein are incorporated by reference in their entireties. In the Figures,

Figure 1 shows a sketch of the 3D cell bioprinting procedure (A). The alginate/gelatin/SaOS- 2 cell suspension is filled into a cartridge, fixed to the printing head (ph). The suspension is pressed through a needle into a culture dish filled with 0.4% CaCl₂ as cross-linking solution.

(B) A bioprinted stack of 13 mm in diameter and 1.5 mm in height, placed in a 24-well plate.

(C) A bioprinted stack after being incubated in medium/FCS for 3 d. (D) SEM image of mineral nodules (no) on the surface of SaOS-2 cells embedded in alginate/gelatin and 100 μΜ polyP^»Ca²⁺-complex and incubated for 3 d in the absence and then for 5 d in the presence of the osteogenic cocktail. The specimen was then inspected by SEM.

Figure 2 shows the morphology of the bioglass preparation used. SEM micrograph. The sintering temperature was 900°C.

Figure 3 shows the SEM images of cell-free printed alginate/gelatin hydrogels. (A) Appearance of the basic alginate/gelatin hydrogel cylinders, containing neither silica nor biosilica nor polyP^»Ca²⁺-complex. (B) Hydrogel containing 10 μg/ml of silicatein. (C and D) Printed basic alginate/gelatin hydrogel containing 50 μΜ ortho-silicate (>si<). (E and F) Cylinders of hydrogel supplemented with 50 μΜ biosilica (>bs<). (G and H) Alginate/gelatin hydrogel cylinders containing 100 μΜ polyP^»Ca²⁺-complex (>polyP<). (I and J) Hydrogel containing both 50 μΜ biosilica (>bs<) and 100 μΜ polyP^»Ca²⁺-complex (>polyP<).

Figure 4 shows the influence of the polymers silica (50 μΜ; hatched rightwards bars), biosilica enzymatically prepared from ortho-silicate and silicatein (50 μΜ; cross hatched white on black), polyP^»Ca²⁺-complex (polyP) (100 μΜ; hatched leftwards), and polyP^»Ca²⁺- complex together with biosilica (polyP + biosilica) (cross hatched black on white) on cell density after an incubation period of 3 d in medium/FCS as determined by MTT assay. The values for the optical density reflect the overall activity of the mitochondrial dehydrogenases and are correlated with the cell number. Control experiments, either containing no polymer (open bars) or silicatein (filled bars), were performed in parallel. The experiments were performed either in the absence (minus bioglass) or presence of 5 mg/ml of bioglass (nano)particles (plus bioglass) in culture medium/FCS. Data represent means ± SD of ten independent experiments (* P < 0.01).

Figure 5 shows the formation of minerals onto SaOS-2 cells. (A) Influence of the tested polymers, polyP^»Ca²⁺-complex (polyP) (100 μΜ; hatched leftwards), silica (50 μΜ; hatched rightwards bars), biosilica (50 μΜ; cross hatched white on black), and polyP^»Ca²⁺-complex together with biosilica (polyP + biosilica) (cross hatched black on white) on the extent of bio mineralization, measured by binding of Alizarin Red S to the inorganic deposits. The color reaction was followed spectroscopically at 570 nm; the values are correlated with the DNA content in the respective sample to allow a direct correlation with the cell numbers. The assays were run in the absence (minus) or presence of 5 mg/ml of bioglass (plus bioglass) in the incubation medium for the organic, printed scaffold. (B to D) Element mapping by EDS spectrometry of the surface of SaOS-2 cells comprising mineral nodules. One nodule is marked (no). Element mapping was performed for O (B), P (C) and C (D). The regions of brighter pseudocolor represent larger accumulations of the respective elements.

Examples

In the following examples, only the inventive method described, using bioglass (bioactive glass) (nano)particles composed of SiC^iCaO^Os of a molar ratio of 55:40:5 with a size of 55 nm and a concentration of 5 mg/ml, as well as a polyP^»Ca²⁺-complex with a polyP chain length of 40 phosphate units and a concentration of 100 μΜ, and ortho-silicate or biosilica with a concentration of 50 μΜ, based on silica monomers. Similar results can be obtained with bioglass (bioactive glass) preparations of a different molar ratio of SiC^CaO^Os and with a smaller or larger size and a lower or higher concentration of the (nano)particles, or with bioglass (bioactive glass) preparations consisting of Si0₂:Na₂0:CaO:P₂05, for example, of a molar ratio of 46.1 :24.4:26.9:2.6 (45S5 Bioglass^®), as well as the presence of lower and higher concentrations of polyP or polyP^»Ca²⁺-complex with lower and higher chain lengths in the alginate/gelatin matrix.

Methods

Polyphosphate

The sodium polyphosphate (Na-polyP of an average chain of 40 phosphate units) used in the Examples has been obtained from Chemische Fabrik Budenheim (Budenheim; Germany). The chelating effect, caused by polyP, can be compensated by mixing with CaCl₂ in a stoichiometric ratio of 2: 1 (polyP:CaCl₂). This salt is termed "polyP^»Ca²⁺-complex". Usually a concentration of 100 μΜ (14 μg/ml polyP^»Ca²⁺-complex) is added to the assays.

Hydrogel preparation

A sodium alginate solution (50 mg/ml), supplemented with 50 mg/ml of low-melting gelatin, is prepared in physiological saline. The gel is supplemented with 10 μΐ/ml of phenol red [0.5%] solution. SaOS-2 cells, growing at the end of the growing phase, are added to the gel at a concentration of 5x10⁵ cells/ml. Where indicated the alginate/gelatin gel is supplemented with 100 μΜ polyP^»Ca²⁺-complex and 50 μΜ ortho-silicate or 50 μΜ biosilica.

Ortho-silicate is prepared from prehydrolyzed TEOS (Tong X, Tang T, Feng Z, Huang B (2002) Preparation of polymer/silica nanoscale hybrids through sol-gel method involving emulsion polymers. II. Poly(ethyl acrylate)/Si0₂. J Appl Polym Sci 86:3532-3536). Biosilica is prepared enzymatically by using, in a reaction volume of 1 mL, 200 μΜ prehydrolyzed TEOS and 20 μg/mL of recombinant silicatein in 50 mM Tris/HCl buffer (pH 7.4; 150 mM NaCl, 0.5% [w/v] PEG [polyethylene glycol]). PEG is added to the reaction in order to harden the biosilica formed. The enzymatic reactions are performed at 22°C for up to 120 min with agitation. Subsequently the reaction mixture is aspirated and the biosilica content determined by using the molybdate assay; the concentration of the molybdate-reactive silica is given in μΜ.

Preparation of bioglass (bioactive glass)

The particles are prepared as described (Hong Z, Liu A, Chen L, Chen X, Jing X (2009) Preparation of bioactive glass ceramic nanoparticles by combination of sol-gel and coprecipitation method. J Non-Crystalline Solids 355:368-372; Hong Z, Reis RL, Mano JF (2009) Preparation and in vitro characterization of novel bioactive glass ceramic nanoparticles. J Biomed Mater Res A 88:304-313). In order to obtain the bioglass nanoparticles with a molar ratio of Si:Ca:P 66:27:7, calcium nitrate and TEOS (Sigma) are dispersed in ethanokwater (1 :2 [mol]). The pH of solution is kept to 1.2 with citric acid (Sigma) and the reaction mixture is stirred until the solution became transparent. Then the solution is added at 55°C, under stirring, to distilled water, ammoniated with ammonium hydrogen phosphate (0.4 g/L). The pH value of the reaction solution is kept at ~ 11. After a period of stirring overnight the precipitate formed is collected by centrifugation and subsequently washed three times with water. Finally, the white bioglass particles are obtained by separation and finally calcination at 900°C. The particle size is approximately 55 nm.

Cells and their incubation conditions

Human osteogenic sarcoma cells, SaOS-2 cells, can be used which are cultivated in McCoy's medium containing 2 mM L-glutamine and gentamycin (50 mg/ml), supplemented with 5% heat-inactivated FCS. For some series of experiments the cells are exposed to the osteogenic cocktail (10 nM dexamethasone, 50 mM ascorbic acid, 5 mM sodium β-glycerophosphate) in order to induce biomineralization.

Where indicated, the added 2.5 mL of medium/FCS around the cylinders, formed of alginate/gelatin/SaOS-2 without or with polyP^»Ca²⁺-complex, silica or biosilica, are supplemented with 5 mg/ml of bioglass (nano)particles. For the determination of the effects on growth incubation lasts 3 d. In order to determine the effect of the different components on the mineralization capacity of SaOS-2 cells the cultures are incubated for 3 d in the absence of the osteogenic cocktail and subsequently for 5 d in the presence of the cocktail.

Microscopic analyses

The scanning electron microscopic (SEM) images can be performed, for example, with a HITACHI SU 8000 (Hitachi High-Technologies Europe GmbH) at low voltage (<1 kV; analysis of near- surface organic surfaces) detector. The SEM mapping experiments (energy dispersive spectrometry [EDS] at sub-micrometer spatial resolution) can be performed, for example, with a XFlash® FlatQUAD (Bruker Nano) using the following settings, 5.5 kV, 260 pA, 120 kcps, 640x480 pixels, 33 nm pixels, 62 min.

Digital light microscopic studies can be performed, for example, using a VHX-600 Digital Microscope (Keyence) equipped with a VH-Z25 zoom lens.

Cell viability and proliferation

The MTT cell viability assay can be applied to determine the cell concentration on the basis of the activities of the dehydrogenase enzymes. After the indicated period of incubation the numbers of SaOS-2 cells are determined after treatment of the alginate/gelatin hydrogel with 55 mM Na-citrate as described (SchloBmacher U, Schroder HC, Wang XH, Feng Q, Diehl- Seifert B, Neumann S, Trautwein A, Muller WEG (2013) Alginate/silica composite hydrogel as a potential morphogenetically active scaffold for three-dimensional tissue engineering. RSC Adv 3: 11185-11194). Subsequently, the cell suspension is incubated with fresh medium containing 100 μΐ of MTT for 12 h in the dark. After removal of the remaining MTT dye 200 μΐ of dimethylsulfoxide (DMSO) are added to solubilize the formazan crystals. Finally the optical densities (OD) at 570 nm are measured using an ELISA reader/spectrophotometer, with background subtraction at 630-690 nm.

Mineralization by SaOS-2 cells in vitro

The mineral deposits onto the cells can be stained with Alizarin Red S. The cells are incubated for 3 d in the absence of the osteogenic cocktail and subsequently for 5 d in the presence of the cocktail. Then the color developed is quantitatively assessed, using the fluorochrome Alizarin Red S as an indicator by applying the spectrophotometric assay (Wiens M, Wang XH, Schroder HC, Kolb U, SchloBmacher U, Ushijima H, Miiller WEG (2010) The role of biosilica in the osteoprotegerin/RANKL ratio in human osteoblast like cells. Biomaterials 31 :7716-7725). The amount of bound Alizarin Red S is given in μιηοΐεβ. Values are normalized to total DNA in the samples. The DNA content can be determined by using the PicoGreen method (Schroder HC, Borejko A, Krasko A, Reiber A, Schwertner H, Miiller WEG (2005) Mineralization of SaOS-2 cells on enzymatically (silicatein) modified bioactive osteoblast-stimulating surfaces. J Biomed Mat Res Part B - Appl Biomater 75B:387-392), with calf thymus DNA as a standard.

Statistical analysis

The results can be statistically evaluated using paired Student's t-test. The generation time of SaOS-2 cells is calculated according to (Powell EO (1956) Growth rate and generation time of bacteria, with special reference to continuous culture. J Gen Microbiol 15:492-511).

Results

Preparation of the bioglass

As solid (nano)particles of bioglass the inventors used the solid glass powder, prepared according to (Hong Z, Liu A, Chen L, Chen X, Jing X (2009) Preparation of bioactive glass ceramic nanoparticles by combination of sol-gel and coprecipitation method. J Non- Crystalline Solids 355:368-372). Using the procedure described under "Material and Methods", the size of the particles was determined to be 55 nm (Fig. 2). The molar ratio of the elements Si0₂:CaO:P₂05 was 55:40:5. By this, the composition of the bioglass is high in phosphorus compared to the one of 45S5 and Bioglass^® (Hench LL (2006) The story of Bioglass^®. J Mater Sci - Mater Med 17:967-978), with 46 mol% Si0₂, 24 mol% Na₂0, and 30 mol% CaO.

Bioprinting of SaOS-2 cells in alginate/gelatin

In the experiments described, the inventors used for 3D cell bioprinting the cell printer "Bioplotter" from Envisiontec.

The SaOS-2 cells were encapsulated into a hydrogel with the components alginate and gelatin, as described under "Methods". The bioprinted cylinders were approximately 300 μιη thick and the parallel oriented patterned layers were arranged in a perpendicular orientation in the consecutive layers (Fig. IB and C).

SEM analyses revealed that the samples containing only alginate/gelatin show an almost homogeneous appearance (Fig. 3 A). Addition of 10 μg/ml of silicatein to the gel does not change the homogeneous pattern (Fig. 3B). However, if 50 μΜ ortho-silicate (final concentration) is added to the hydrogel the samples comprise clusters of 500 nm-sized silica drops (Fig. 3C and D). The clusters and patches of biosilica (50 μΜ) within the hydrogel are widespread (Fig. 3E and F). Addition of 100 μΜ polyP^»Ca²⁺-complex to the hydrogel, prior to the bioprinting process, revealed the appearance of crystal-like precipitates in the hydrogel cylinders (Fig. 3G and H). The size of those deposits range between 10 and 15 μιη. If the hydrogel is supplemented both with polyP^»Ca²⁺-complex and biosilica the characteristic deposits, crystal-like for polyP^»Ca²⁺-complex and round-shaped clusters of drops for biosilica become obvious (Fig. 31 and J).

Effect of polyP, silica and biosilica on growth of SaOS-2 cells in the alginate/gelatin hydrogel In the absence of the bioglass in the medium the two natural polymers, polyP^»Ca²⁺-complex and biosilica, but also to a smaller extent ortho-silica, display a significant stimulation of cell growth, if the cell density is measured after an incubation period of 3 d (Fig. 4). Addition of 100 μΜ polyP^»Ca²⁺-complex caused a significant increase in cell density from 0.52±0.06 optical density units (controls without any of those polymers) to 1.38±0.21 units. Addition of 50 μΜ biosilica to the hydrogel resulted in an increase to 1.63±0.22 units; co-addition of 100 μΜ polyP^»Ca²⁺-complex and 50 μΜ biosilica did not change the cell density significantly. This might imply that under these conditions the maximal potency of the cells to proliferate has been reached. During the 3 d incubation period the cells underwent 1.6 doublings. If 50 μΜ of chemically polycondensed silica, as ortho-silicate, is added to the hydrogel the cell growth increased to a smaller extent, but still significantly, to 0.87±0.11 units. In contrast, 10 μg/ml of silicatein added alone without ortho-silicate substrate to the hydrogel, had no significant effect on the cell density (Fig. 4). However, if silicatein (10 μg/ml) was added together with 50 μΜ ortho-silicate to the alginate/gelatin hydrogel the SaOS-2 cells proliferated and reached a cell density of 1.48±0.27 units (data not included in Fig. 4). This result indicates that also in the hydrogel environment silicatein, if added together with its substrate ortho-silicate, has the capacity to form enzymatically biosilica.

Addition of 5 mg/ml of bioglass (nano)particles to the cells embedded into alginate/gelatin hydrogel, containing in separate series of experiments, the polymers polyP^»Ca²⁺-complex, silica, biosilica or polyP^»Ca²⁺-complex together with biosilica did not significantly change the growth potency of the cells (Fig. 4); the extent of the cell densities was very similar to those seen in the assays lacking bioglass.

Influence of the polymers on biomineralization

The SaOS-2 cells embedded into alginate/gelatin hydrogel, supplemented with the different polymers examined here, were incubated for 3 d in the absence of the osteogenic cocktail and then for additional 5 d in the presence of the cocktail. The extent of biomineral formation was determined spectrophotometrically and the values were correlated with the DNA amount in the respective sample. By that, a correlation of the magnitude of mineral deposits on the SaOS-2 cells could be quantified. As summarized in Fig. 5A, the amount of Alizarin Red S- positive reaction, in the assay without added bioglass, increased significantly from 0.34±0.05 nmoles of Alizarin Red S formedVg DNA to 0.73±0.12 nmoles^g (for 100 μΜ polyP^»Ca²⁺- complex), to 0.61 ±0.10 nmoles^g (50 μΜ silica), to 0.91 ±0.13 nmoles^g (50 μΜ biosilica) and to 1.38±0.29 nmoles^g (100 μΜ polyP^»Ca²⁺-complex together with 50 μΜ biosilica); the values in the controls without any polymer and with 10 μg/ml silicatein vary around 0.35 nmoles^g. Addition of 5 mg/ml of bioglass to the incubation assay increased the extent of mineralization significantly, for polyP^»Ca²⁺-complex to 1.31±0.14 nmoles^g, for silica to 0.98±0.14 nmoles g, for biosilica to 1.62±0.19 nmoles^g and for the two components together (polyP^»Ca²⁺-complex and biosilica) to 2.31±0.43 nmoles^g (Fig. 5). Likewise, also in the absence of any additional polymer the potency of the embedded SaOS-2 increased significantly from 0.34±0.05 nmoles^g DNA, in the absence of bioglass in the culture medium, to 0.61±0.09 nmoles^g in the presence of bioglass.

Crystallite formation onto SaOS-2 cells

An incubation period of 5 d of the cells in medium/FCS, supplemented with osteogenic cocktail resulted in the formation of mineral deposits. Those 1 to 10 μιη large deposits can be visualized by SEM (Fig. 1 D). EDS spectrometric analysis at sub-micrometer spatial resolution revealed an accumulation of the signals for the elements O, P and C (Fig. 5B-D); the element Ca is likewise present at higher concentrations at the mineral nodules (not shown). This finding indicates that the deposits are composed of Ca-phosphate, but also of considerable high amounts of Ca-carbonate.

Claims

1. A method for the preparation of bioglass (bioactive glass) scaffolds bone implants comprising bone cells, comprising or consisting of

i) embedding said bone cells into a hydrogel,

ii) 3D-bioprinting of said resulting cell hydrogel suspension,

and

iii) adding a cell- free suspension of (nano)particles of bioglass (bioactive glass) to said cell hydrogel suspension in parallel or after bioprinting, whereby an outer bioglass-containing matrix surrounding an inner cell-compatible matrix is formed.

2. The method according to claim 1, wherein said hydrogel is selected from an alginate hydrogel, an alginate/gelatin hydrogel, and an alginate hydrogel containing another collagen- derived product.

3. The method according to claim 1 or 2, wherein said cell hydrogel suspension is supplemented with a morphogenetically active oligomer or polymer.

4. The method according to claim 3, wherein said morphogenetically active oligomer or polymer is selected from inorganic pyrophosphate, oligoP, polyP, and a complex of these oligomers/polymers with divalent metal ions.

5. The method according to claim 4, wherein said divalent metal ions are calcium ions [polyP^»Ca²⁺-complex] .

6. The method according to claim 3, wherein said morphogenetically active polymer is polymeric silicic acid (silica) or one of its salts.

7. The method according to any of claims 3 to 6, wherein said morphogenetically active oligomer or polymer is a combination of inorganic pyrophosphate, oligoP or polyP, or a complex of these oligomers/polymers with divalent metal ions, with polymeric silicic acid (silica) or its salts.

8. The method according to claim 6 or 7, wherein said polymeric silicic acid has been formed by an enzyme or protein involved in biosilica (amorphous, hydrated silicon oxide) metabolism, such as silicatein or a silicatein fusion protein or combinations thereof.

9. The method according to any of claims 6 to 8, wherein silicatein or a silicatein fusion protein or combinations thereof, as well as a suitable substrate are present.

10. The method according to claim 9, wherein said silicatein polypeptide or silicatein fusion protein has been produced using a prokaryotic or eukaryotic expression system, or has been produced synthetically.

11. The method according to any of claims 3 to 6, comprising a combination of inorganic pyrophosphate, oligoP or polyP, or a complex of these oligomers/polymers with divalent metal ions, with monomeric silicic acid (orthosilicic acid).

12. The method according to any of claims 1 to 11, wherein said bioglass (bioactive glass) (nano)particles are composed of SiC^iCaO^Os or Si02:Na₂0:CaO:P205 of various molar ratios, for example Si0₂:CaO:P₂0₅ of a molar ratio (mol.%) of 55 :40:5 or Si0₂:Na₂0:CaO:P₂0₅ of a molar ratio (mol.%) of 46.1 :24.4:26.9:2.6 (45S5 Bioglass^®).

13. The method according to any of claims 1 to 12, wherein said suspension of bioglass (bioactive glass) (nano)particles is printed in parallel with the cell-containing hydrogel using a three-dimensional (3D) printing technique (two-component scaffold).

14. The method according to any of claims 1 to 12, wherein said suspension of bioglass (bioactive glass) (nano)particles is mixed with the cell-containing hydrogel before 3D bioprinting of the scaffold.

15. A 3D-bioprinted bioglass (bioactive glass) scaffold bone implant material comprising bone cells, obtained by a method according to any of claims 1 to 13.

16. The bone implant material according to claim 15 for use in the treatment of bone defects.

17. The bone implant material for use according to claim 16 in the form of customized, morphogenetically active and biodegradable implants produced by a rapid prototyping/solid free-form fabrication method.