Sponge comprising a recombinant collagen-like peptide (CLP) and bioactive glass

Abstract

The present invention relates to a method of preparing a bioactive glass containing sponge based on collagen-like protein comprising or consisting of the steps: i) providing an aqueous solution comprising at least one collagen-like protein, at least one bioactive glass and optionally at least one additive; ii) performing a lyophilization step to obtain a sponge; iii) thereafter performing a dehydrothermal treatment to the sponge obtained in step ii); and iv) optionally sterilizing the sponge obtained after step iii). Furthermore, the present invention pertains to a sponge obtained by the method according to the present invention. Moreover, the present invention refers to particles obtained by shredding, milling or fragmentation of the sponge according to the present invention. Finally, the present invention refers to the use of the sponge according to the present invention or the particles according to the present invention for bone regeneration, cartilage repair, skin repair or as dental membrane.

Description

[0001] Sponge comprising a recombinant collagen-like peptide (CLP) and bioactive glass

[0002] Field of the invention

[0003] The present invention relates to a method of preparing a bioactive glass containing sponge based on collagen-like protein comprising or consisting of the steps: i) providing an aqueous solution comprising at least one collagen-like protein, at least one bioactive glass and optionally at least one additive; ii) performing a lyophilization step to obtain a sponge; iii) thereafter performing a dehydrothermal treatment to the sponge obtained in step ii); and iv) optionally sterilizing the sponge obtained after step iii).

[0004] Furthermore, the present invention pertains to a sponge obtained by the method according to the present invention. Moreover, the present invention refers to particles obtained by shredding, milling or fragmentation of the sponge according to the present invention. Finally, the present invention refers to the use of the sponge according to the present invention or the particles according to the present invention for bone regeneration, cartilage repair, skin repair or as dental membrane.

[0005] Background

[0006] Animal collagen derived compositions containing calcium phosphate used for bone regeneration are known in the art.

[0007] For example, US 20100196489 A1 describes the process for a paste or putty made from a synthetic bone growth composition comprising a fibrillar collagen and a calcium phosphate component. The process includes an intermediate sponge manufactured by lyophilization and stabilization by dehydrothermal treatment. In detail, fibrillar bovine collagen isolated from dermis was mixed with calcium hydrogen phosphate in 30 mM HCI. After pouring, the constructs were frozen at -80 °C for two hours and lyophilized thereafter. Freeze-dried constructs were cross-linked by dehydrothermal treatment in vacuo at 110 °C for 48 hours. Cross-linked sponges were milled to particles and applied to manufacture a paste.

[0008] Hashimoto, Y.; et al.: Evaluation of Bone Regeneration with a Collagen Model Polypeptides / Alpha- Tricalcium Phosphate Sponge. Journal of Oral and Maxillofacial Surgery, (September 2014) Vol. 72, No. 9, Supp. SUPPL. 1 , pp. e184-e185 disclose a dehydrothermally stabilized sponge made from a- tricalcium phosphate and a combination with a collagen model polypeptide consisting of a Pro-Hyp- Gly sequence. This Poly(PHG) was synthesized by direct polycondensation of (Pro-Hyp-Gly)n with 1-hydroxybenzotriazole and 1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide hydrochloride. For the sponge preparation, porous a-TCP particles were mixed with an aqueous poly(PHG) solution at a concentration of 10 mg / ml. The mixture was then poured into plastic molds, and all samples were immediately frozen to -80 °C and freeze-dried for 24 hours. The freeze-dried constructs were subsequently cross-linked in vacuo at 140 °C for 10 hours. The poly(PHG) / a-TCP sponges were sterilized with ethylene oxide gas at 40°C.

[0009] There is a need to replace calcium phosphate in such compositions in order to obtain improved bioactivity. Furthermore, it is desired to replace animal derived compounds, like collagen.

[0010] The object of the present invention was to provide a non-animal derived composition having similar structural characteristics compared to collagen compositions comprising calcium phosphate.

[0011] In this regard the inventors of the present invention surprisingly found that the sponges according to the present invention based on collagen-like protein and containing bioactive glass can solve this object. Bioactive glass contains four oxides (silicon oxide, sodium oxide, calcium oxide and phosphor oxide) essential for the human body makes it highly biocompatible. Aqueous suspension of bioactive glass shows anti-bacterial, anti-oxidative and anti-inflammatory effects. Upon activation with fluid, bioactive glass releases soluble silica and calcium ions that stimulate osteoprogenitor cells at genetic level. SiO2-based bioactive glass can induce the formation of surface-active carbonated hydroxyapatite (HA) layers, increasing rapidly bone bonding. Furthermore, bioactive glass offers excellent absorption properties or can be applied as a carrier for liquids and active substances.

[0012] However, the standard solvents for animal collagens are acetic acid or hydrochloric acid that result in a viscous solution. Despite being soluble at acidic conditions, animal collagens exhibit no solubility at neutral or alkaline pH. However, bioactive glass powders immersed in distilled water led to an immediate rise of the pH to 10. This fast pH increase hinders the solubilization of the animal collagens and thereby prevents a lean processing including simultaneous mixture of bioactive glass with animal collagens.

[0013] In addition, the obtained sponges also show swelling after being contacted with a liquid that is not observed for sponges based on pure collagen-like peptide. Furthermore, the sponges according to the present invention show improved non-destructive compression, as well as in vitro bone cell growth. Moreover, the present process is as well improved in view of the known processes for sponges derived from animal collagen by requiring less process steps.

[0014] Summary of the invention

[0015] Therefore, in a first aspect, the present invention refers to a method of preparing a bioactive glass containing sponge based on collagen-like protein comprising or consisting of the steps: i) providing an aqueous solution comprising at least one collagen-like protein, at least one bioactive glass and optionally at least one additive; ii) performing a lyophilization step to obtain a sponge; iii) thereafter performing a dehydrothermal treatment to the sponge obtained in step ii); and iv) optionally sterilizing the sponge obtained after step iii).

[0016] In a second aspect, the present invention pertains to a sponge obtained by the method according to the present invention.

[0017] In a third aspect, the present invention refers to particles obtained by shredding, milling or fragmentation of the sponge according to the present invention.

[0018] Finally, in a fourth aspect, the present invention refers to the use of the sponge according to the present invention for bone regeneration, cartilage repair, skin repair or as dental membrane.

[0019] Description of the figures

[0020] Fig. 1 : Photos of sponges made from collagen-like protein and bioactive glass. For a proof-of- concept, (A) 20 mg / ml and (B) 40 mg / ml collagen-like protein were mixed with 20 mg / ml bioactive glass in water to manufacture composite sponges. The applied bioactive glass has a D50 value of 3.86 pm. The prepared dispersion was lyophilized and dehydrothermally stabilized to form stable dry sponges. After overnight incubation in water sponges made from (C) 20 mg / ml and (D) 40 mg / ml collagen-like protein plus 20 mg / ml bioactive glass showed integrity and swelling. Form stability was concentration dependent: sponges made from (E) 20 mg / ml collagen-like protein resulted in deformable composites, whereas sponges made from (F) 40 mg / ml collagen-like protein showed form stability.

[0021] Fig. 2: The in this invention described process for sponges made from collagen-like protein with bioactive glass has been tested for its transferability to rat tail collagen. In detail, 0.1 % acetic acid was applied as the standard solvent of rat tail collagen to directly mix with the two components - bioactive glass and rat tail collagen. Here, two concentrations of (A) 5 mg / ml and (B) 45 mg / ml bioactive glass were mixed with 5 mg / ml rat tail collagen. In both setups, the rat tail collagen was not dissolved after overnight incubation at room temperature on a shaker. (C) Applying few drops of the solution on pH-testing stripes confirmed a pH increase from 3 to 10 or 11 induced by the supplementation of bioactive glass. This proof-of-concept showed that a direct process transfer to animal collagens is not possible.

[0022] Fig. 3: Composite sponges were hydrated in phosphate-buffered saline for 24 hours. (A) Three different compositions (pictures from left to right) made of (I) 25 mg / ml collagen-like protein plus 25 mg / ml bioactive glass, (II) 50 mg / ml collagen-like protein plus 50 mg / ml bioactive glass, and (III) 75 mg / ml collagen-like protein plus 75 mg / ml bioactive glass were tested for hydration characteristics, including fluid uptake, and swelling. (B) Integrity and swelling behavior were clearly visible after hydration. Fig. 4: Total fluid uptake was measured by weight gain after incubation for 24 hours in phosphate- buffered saline. Mean values and standard deviation were calculated from triplicates (n = 3).

[0023] Fig. 5: Fluid absorption decreased with increasing formulation concentration. Fluid absorption was calculated relatively by comparing weight of the hydrated sponges to respective dry weight. Plotted mean values include three independent samples (n - 3).

[0024] Fig. 6: Swelling was quantified by diameter change after hydration for 24 hours in phosphate- buffered saline. An increasing swelling behavior was shown with the concentration of components. All quantitative assessment were performed in triplicates (n - 3) and standard deviation was calculated.

[0025] Fig. 7: Sponges were compressed by a mechanical testing device. Recorded stress to strain correlations were applied to derive the slope of the elastic range, describing the Young’s moduli. Mean values were calculated for the compression of five samples.

[0026] Fig. 8: After compression sponges showed clear imprints of the probe (visualized by an arrow), but shape was maintained, and no breakage was observed. Here exemplary shown for the composition made of 75 mg / ml collagen-like protein and 75 mg / ml bioactive glass.

[0027] Fig. 9: Microstructure was analyzed by scanning electron microscopic imaging of cryo cross sections. Three different compositions (pictures from left to right) made of (I) 25 mg / ml collagen-like protein plus 25 mg / ml bioactive glass, (II) 50 mg / ml collagen-like protein plus 50 mg / ml bioactive glass, and (III) 75 mg / ml collagen-like protein plus 75 mg / ml bioactive glass were applied for this structure analysis. (A) Pore structure and size changed with concentration of the formulation. (B) All formulations showed well integrated particles within the formed scaffold walls.

[0028] Fig. 10: The pore size was quantified by an image analysis of the captured scanning electron microscopic images. Here for, the size of 20 pores was measured and mean values were derived.

[0029] Fig. 11: Integrity and homogeneity of composites have limitations. (A) A formulation of 25 mg / ml collagen-like protein loaded with 75 mg / ml bioactive glass showed (I) a clear separation in an organic phase and an inorganic phase. (II) Shape of the dry formulation was lost (III) after hydration. (B) A higher loading ratio of 150 mg / ml bioactive glass to 25 mg / ml collagen-like protein completely destabilizes (I) the apparent integrity of the dry composite (II) after fluid contact.

[0030] Fig. 12: Sponges were seeded with Cal-72 bone cells and cultured for 7 days before staining with MTT dye to visualize viable cells and area of cell growth. (A) A formulation with 20 mg / ml collagen- like protein and 20 mg / ml bioactive glass was compared with a (B) plain collagen-like protein sponge (as well 20 mg / ml). (I) Top views and (II) cross-sections of MTT-stained scaffolds were photographically captured. Detailed description

[0031] Numerical ranges that are indicated in the format “from x to y” also include the stated values. If several preferred numerical ranges are indicated in this format, it is self-evident that all ranges that result from the combination of the various endpoints are also included.

[0032] "One or more", as used herein, relates to at least one and comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9 or more of the referenced species. Similarly, "at least one" means one or more, i.e. 1 , 2, 3, 4, 5, 6, 7, 8, 9 or more. "At least one", as used herein in relation to any component, refers to the number of chemically different molecules, i.e. to the number of different types of the referenced species, but not to the total number of molecules. For example, "at least bioactive glass" means that at least one type of molecule falling within the definition for a bioactive glass is used but that also two or more different types of bioactive glass falling within this definition can be present but does not mean that only one or more molecules of one type of bioactive glass are present.

[0033] All percentages given herein in relation to the compositions or formulations relate to wt-% relative to the total weight of the respective composition, if not explicitly stated otherwise.

[0034] “Essentially free of’ according to the present invention with regard to compounds means that the compound can only be present in an amount, which does not influence the characteristics of the composition, in particular the respective compound is present in less than 3 wt.-%, preferably 1 wt.- %, more preferably 0.01 wt.-%, based on the total weight of the composition or is not present at all.

[0035] The weight average molecular weight Mw and the number average molecular weight Mn can be determined by GPC employing polystyrene standards.

[0036] Therefore, in a first aspect, the present invention refers to a method of preparing a bioactive glass containing sponge based on collagen-like protein comprising or consisting of the steps: i) providing an aqueous solution comprising at least one collagen-like protein, at least one bioactive glass and optionally at least one additive; ii) performing a lyophilization step to obtain a sponge; iii) thereafter performing a dehydrothermal treatment to the sponge obtained in step ii); and iv) optionally sterilizing the sponge obtained after step iii).

[0037] In a second aspect, the present invention pertains to a sponge obtained by the method according to the present invention.

[0038] In a third aspect, the present invention refers to particles obtained by shredding, milling or fragmentation of the sponge according to the present invention. Finally, in a fourth aspect, the present invention refers to the use of the sponge according to the present invention for bone regeneration, cartilage repair, skin repair or as dental membrane.

[0039] These and other aspects, embodiments, features, and advantages of the invention will become apparent to a person skilled in the art through the study of the following detailed description and claims. Any feature from one aspect of the invention can be used in any other aspect of the invention. Furthermore, it will readily be understood that the examples contained herein are intended to describe and illustrate the invention but not to limit the invention and that, in particular, the invention is not limited to these examples.

[0040] In the method at least one collagen-like protein is used. In general, all collagen-like proteins are suitable.

[0041] In a preferred embodiment of the present invention the collagen-like protein is a collagen-like protein from Streptococcus pyogenes, which is preferably the Scl2 protein from Streptococcus pyogenes.

[0042] Expression of collagen-like proteins have been attempted in several systems, including Escherichia coli and Saccharomyces cerevisiae. In one embodiment the at least one collagen-like protein is a bacterial collagen-like protein, preferably produced by fermentation in Pichia, Brevibacillus, Bacillus, Escherichia or Corynebacterium, preferably Pichia pastoris, Brevibacillus choshinensis or Corynebacterium glutamicum.

[0043] In a preferred embodiment the collagen-like proteins may be expressed in Corynebacterium, preferably in Corynebacterium glutamicum.

[0044] One particularly suitable collagen-like protein is derivable from following polynucleotide.

[0045] A polynucleotide encoding an amino acid sequence that is at least > 60%, identical to the amino acid sequence of SEQ ID NO:1 , wherein the polynucleotide is a replicable polynucleotide encoding a collagen-like protein and wherein the amino acid sequence comprises a deletion of at least 38 amino acids at the N-terminus of the amino acid sequence of SEQ ID NO:1.

[0046] It is preferred, when the amino acid sequence comprises a deletion of between 38 and 74 amino acids at the N-terminus of the amino acid sequence of SEQ ID NO: 1. This includes a complete deletion of the N-terminal V-domain (comprising 74 amino acids) and different truncations of the V- domain of at least 38 amino acids.

[0047] In a preferred embodiment, the amino acid sequence that is at least > 60%, identical to the amino acid sequence of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

[0048] In a further configuration, the amino acid sequence that is at least > 65%, or > 70%, or > 75%, or > 80%, or a 85% identical to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4. In a preferred configuration, the polynucleotide encodes an amino acid sequence that is at least > 90%, s 92%, s 94%, 96%, s 97%, s 98%, s 99% or 100%, preferably 97%, particularly preferably s 98%, very particularly preferably 99%, and extremely preferably 100%, identical to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

[0049] In a preferred embodiment of the present invention the polynucleotide is a replicable nucleotide sequence encoding the collagen-like protein from Streptococcus pyogenes.

[0050] Polynucleotide and nucleic acid molecules comprising such sequences and encoding polypeptide variants of SEQ ID NO:1 to 4, which contain one or more insertion(s) or deletion(s) are suitable as well. Preferably, the polypeptide contains a maximum of 5, a maximum of 4, a maximum of 3, or a maximum of 2, insertions or deletions of amino acids.

[0051] Mixture of polypeptides comprising one of the polypeptide variants of SEQ ID NO:1 to 4 and on or more of the truncated variants of the collagen-like protein of SEQ ID NO:5 to 12 can be used as well.

[0052] Plasmids and vectors that comprise the nucleotide sequences according to the invention and optionally replicate in microorganisms of the genera Pichia, Corynebacterium, Pseudomonas or Escherichia are suitable. In a preferred configuration, the vector comprising the nucleotide sequences according to the present invention is suitable for replication in yeast of the genus Pichia pastoris.

[0053] Microorganisms of the genera Pichia, Corynebacterium, Pseudomonas or Escherichia that comprise the polynucleotides, vectors and polypeptides according to the invention are suitable as well. Preferred microorganisms are Pichia pastoris, Brevibacillus choshinensis or Corynebacterium glutamicum.

[0054] Microorganism of the species P. pastoris, E. coli, P. putida or C. glutamicum comprising any of the nucleotide sequences according to the present invention any of the polypeptides or any of the vectors according to the present invention are suitable.

[0055] The microorganism may be a microorganism in which the nucleotide sequence is present in overexpressed form.

[0056] Overexpression according to the invention means, generally, an increase in the intracellular concentration or activity of a ribonucleic acid, a protein (polypeptide) or an enzyme, compared with the starting strain (parent strain) or wild-type strain, if this is the starting strain. A starting strain (parent strain) is taken to mean the strain on which the measure leading to the overexpression was carried out.

[0057] In the overexpression, the methods of recombinant overexpression are preferred. These include all methods in which a microorganism is produced using a DNA molecule provided in vitro. Such DNA molecules comprise, for example, promoters, expression cassettes, genes, alleles, encoding regions etc. These are converted into the desired microorganism by methods of transformation, conjugation, transduction or like methods.

[0058] The extent of the expression or overexpression can be established by measuring the amount of the mRNA transcribed by the gene, by determining the amount of the polypeptide, and by determining the enzyme activity.

[0059] The bacterial collagen-like protein can be obtained in a fermentative process comprising the following steps: a) fermentation of a microorganism according to the present invention in a medium, b) accumulation of the bacterial collagen-like protein in the medium, wherein a fermentation broth is obtained.

[0060] The culture medium or fermentation medium that is to be used must appropriately satisfy the demands of the respective strains. Descriptions of culture media of various microorganisms are contained in the handbook "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington D.C., USA, 1981 ). The terms culture medium and fermentation medium or medium are mutually exchangeable.

[0061] As carbon source, sugars and carbohydrates can be used, such as, e.g., glucose, sucrose, lactose, fructose, maltose, molasses, sucrose-containing solutions from beet sugar or sugar cane processing, starch, starch hydrolysate and cellulose, oils and fats, such as, for example, soybean oil, sunflower oil, groundnut oil and coconut fat, fatty acids, such as, for example, palmitic acid, stearic acid and linoleic acid, alcohols such as, for example, glycerol, methanol and ethanol, and organic acids, such as, for example, acetic acid or lactic acid.

[0062] As nitrogen source, organic nitrogen compounds such as peptones, yeast extract, meat extract, malt extract, corn-steep liquor, soybean meal and urea or inorganic compounds such as ammonium sulphate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate can be used. The nitrogen sources can be used individually or as a mixture.

[0063] As phosphorus source, phosphoric acid, potassium dihydrogenphosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts can be used.

[0064] The culture medium must, in addition, contain salts, for example in the form of chlorides or sulphates of metals such as, for example, sodium, potassium, magnesium, calcium and iron, such as, for example, magnesium sulphate or iron sulphate, which are necessary for growth. Finally, essential growth substances such as amino acids, for example homoserine and vitamins, for example thiamine, biotin or pantothenic acid, can be used in addition to the above-mentioned substances.

[0065] Said starting materials can be added to the culture in the form of a single batch or supplied in a suitable manner during the culturing.

[0066] Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water, or acid compounds such as phosphoric acid or sulphuric acid, are used in a suitable manner for pH control of the culture. The pH is generally adjusted to 6.0 to 8.5, preferably 6.5 to 8. For control of foam development, antifoams can be used, such as, for example, polyglycol esters of fatty acids. For maintaining the stability of plasmids, suitable selectively acting substances such as, for example, antibiotics, can be added to the medium. The fermentation is preferably carried out under aerobic conditions. In order to maintain said aerobic conditions, oxygen or oxygen-containing gas mixtures such as, for example, air, are introduced into the culture. The use of liquids that are enriched with hydrogen peroxide is likewise possible. Optionally, the fermentation is carried out at superatmospheric pressure, for example at a superatmospheric pressure of 0.03 to 0.2 MPa. The temperature of the culture is usually 20°C to 45°C, and preferably 25°C to 40°C, particularly preferably 30°C to 37°C. In the case of batch or fed-batch processes, the culturing is preferably continued until an amount sufficient for the measure of obtaining the desired organic chemical compound has formed. This goal is usually reached within 10 hours to 160 hours. In continuous processes, longer culture times are possible. Due to the activity of the microorganisms, enrichment (accumulation) of the fine chemicals in the fermentation medium and / or in the cells of the microorganisms occurs.

[0067] Examples of suitable fermentation media may be found, inter alia, in patent documents US 5,770,409, US 5,990,350, US 5,275,940, WO 2007 / 012078, US 5,827,698, WO 2009 / 043803, US 5,756,345 or US 7,138,266; appropriate modifications may optionally be carried out to the requirements of the strains used.

[0068] The process may be characterized by a process which is selected from the group consisting of batch process, fed-batch process, repetitive fed-batch process and continuous process.

[0069] The process may be further characterized by a fine chemical, or a liquid, or a solid fine chemicalcontaining product is obtained from the fine chemical-containing fermentation broth.

[0070] The performance of the processes or fermentation processes according to the invention with respect to one or more of the parameters selected from the group of concentration (compound formed per volume), yield (compound formed per carbon source consumed), volumetric productivity (compound formed per volume and time) and biomass-specific productivity (compound formed per cell dry mass or bio dry mass and time or compound formed per cell protein and time) or other process parameters and combinations thereof, is increased by at least 0.5%, at least 1 %, at least 1.5% or at least 2%, based on processes or fermentation processes with microorganisms in which the promoter variant according to the invention is present.

[0071] Owing to the measures of the fermentation, a fermentation broth is obtained which contains the desired collagen-like protein, and preferably amino acid or organic acid.

[0072] Then, a product in liquid or solid form that contains the collagen-like protein is provided or produced or obtained.

[0073] A fermentation broth means, in a preferred embodiment, a fermentation medium or nutrient medium in which a microorganism was cultured for a certain time and at a certain temperature. The fermentation medium, or the media used during the fermentation, contains / contain all substances or components that ensure production of the desired collagen-like protein and typically ensure growth and / or viability.

[0074] On completion of the fermentation, the resultant fermentation broth accordingly contains a) the biomass (cell mass) of the microorganism resulting from growth of the cells of the microorganism, b) the desired collagen-like protein formed in the course of the fermentation, c) the organic by-products possibly formed in the course of the fermentation, and d) the components of the fermentation medium used, or of the starting materials, that are not consumed by the fermentation, such as, for example, vitamins such as biotin, or salts such as magnesium sulphate.

[0075] The organic by-products include substances which are generated in addition to the respective desired compound by the microorganisms used in the fermentation and are possibly secreted.

[0076] The fermentation broth is withdrawn from the culture vessel or the fermentation container, optionally collected, and used for providing a product in liquid or solid form containing the collagen-like protein. The expression "obtaining the collagen-like protein-containing product" is also used therefor. In the simplest case, the collagen-like protein-containing fermentation broth withdrawn from the fermentation container is itself the product obtained.

[0077] By way of one or more of the measures selected from the group a) partial (> 0% to < 80%) to complete (100%) or virtually complete (2 80%, > 90%, 2 95%, 2 96%, 2 97%, > 98%, > 99%) removal of the water, b) partial (> 0% to < 80%) to complete (100%) or virtually complete (> 80%, > 90%, > 95%, > 96%, 2 97%, > 98%, 2 99%) removal of the biomass, wherein this is optionally inactivated before the removal, c) partial (> 0% to < 80%) to complete (100%) or virtually complete (2 80%, 2 90%, 2 95%, 2 96%, 2 97%, 2 98%, 2 99%, 2 99.3%, 2 99.7%) removal of the organic by-products formed in the course of the fermentation, and d) partial (> 0%) to complete (100%) or virtually complete (2 80%, 2 90%, 2 95%, 2 96%, 2 97%, 298%, 299%, 2 99.3%, 299.7%) removal of the components of the fermentation medium used or the starting materials that are not consumed by the fermentation, a concentration or purification of the desired collagen-like protein is achieved from the fermentation broth. In this manner, products are isolated that have a desired content of the compound.

[0078] The partial (> 0% to < 80%) to complete (100%) or virtually complete (2 80% to < 100%) removal of the water (measure a)) is also termed drying.

[0079] In a variant of the process, by complete or virtually complete removal of the water, the biomass, the organic by-products and the non-consumed components of the fermentation medium used, pure ( 80% by weight, > 90% by weight) or high-purity (> 95% by weight, s 97% by weight, > 99% by weight) product forms of the desired collagen-like protein, preferably bacterial collagen-like protein, are successfully arrived at. For the measures according to a), b), c) or d), a great variety of technical instructions are available in the prior art.

[0080] In the case of processes for producing bacterial collagen-like protein processes are preferred in which products are obtained that do not contain any components of the fermentation broth. These products are used, in particular, in human medicine, in the pharmaceuticals industry, and in the food industry.

[0081] In the method according to the present invention the collagen like protein can preferably be present in the aqueous solution with a concentration range from 2.5 to 200 mg / ml.

[0082] In the method according to the present invention at least one bioactive glass is present.

[0083] In the present invention the term "bioactive glass" means an inorganic glass material having an oxide of silicon as its major component and which is capable of bonding with growing tissue when reacted with physiological fluids.

[0084] Bioactive glasses are well known to the skilled person, and are disclosed, for example, in: L. Hench and J. Wilson “An Introduction to Bioceramics” , World Scientific, New Jersey (1993), the contents of which are hereby incorporated by reference. In the present invention any known bioactive glass is in general suitable.

[0085] The bioactive glass preferably includes between 40 and 86 wt.-% of silicon dioxide oxide (SiOz), between about 0 and 35 wt.-% of sodium oxide (Na2O), between about 4 and 46 wt.-% calcium oxide (CaO), and between about 1 and 15 wt.-% phosphorus oxide (P2O5). More preferably, the glass includes between 40 and 60 wt.-% of silicon dioxide oxide (SiOa), between about 5 to 30 wt-% of sodium oxide (NazO), between about 10 and 35 wt.-% calcium oxide (CaO), and between about 1 and 12 wt-% phosphorus oxide (P2O5). The oxides can be present as solid solutions or mixed oxides, or as mixtures of oxides. The wt.-% is based on the total weight of the bioactive glass.

[0086] CaFz, B2O3, AI2O3, MgO and K2O may be included in the composition in addition to silicon, sodium, phosphorus and calcium oxides. The preferred range for B2O3 is between 0.01 and 10 wt.-%. The preferred range for K2O is between 0 and 8 wt-%. The preferred range for MgO is between 0.01 and 5 wt-%.

[0087] Anti-microbial salts such as AgNOs, CuO, and ZnO, or other antimicrobial salts of the silver, copper and zinc ions, such as nitrates, and acetates can be added. The preferred range for these salts is between 0.01 and 5 wt.-%.

[0088] One suitable and commercially available bioactive glass is Bioglass® (a trademark of University of Florida), which has a composition including about 45 wt.-% silicon dioxide, about 24.5 wt.-% sodium oxide, about 6 wt.-% phosphorus oxide, and about 24.5 wt.-% calcium oxide. In one embodiment the bioactive glass is a calcium sodium phosphosilicate powder.

[0089] In one embodiment the bioactive glass is composed of particles with a D50 value of 1 to 5 pm, preferably 3.9 pm and a D99 value of 10 to 20 pm, preferably 14.5 pm.

[0090] In one embodiment the bioactive glass consists of 24.5±3 wt% CaO, 24.5±3 wt% Na2O, 6±2 wt% P2O5, 45±3 wt% SiO2.

[0091] Particulate, non-interlinked bioactive glass is preferred in the present invention. That is, the glass is in the form of small, discrete particles, rather than a fused matrix of particles or a mesh or fabric (woven or non-woven) of glass fibers. Note that under some conditions the discrete particles of the present invention may tend to cling together because of electrostatic or other forces but are still considered to be non- interlinked. The particles can be contained in powder form. Preferably the particle size is less than about 90 microns; more preferably 2 to 50 microns, most preferably 4 to 10 microns, as measured by SEM, sieving or laser light scattering techniques.

[0092] The bioactive glass can be highly porous bioactive glass. When highly porous bioactive glass is used in place or in addition to small particles of bioactive glass, the pore size is between about 0 and 500 pm, preferably between about 10 and 150 pm, and more preferably, between about 50 and 100 pm. The degree of porosity of the bioactive glass is preferably about 0 to 85 %, more preferably about 30 to 80 %, and most preferably about 40 to 60 %. Porous bioactive glass can be prepared, for example, by incorporating a leachable substance into the bioactive glass composition and leaching the substance out of the glass. Suitable leachable substances are well known to the skilled person, and include, for example, sodium chloride and other water-soluble salts. The particle size of the leachable substance is roughly the size of the resulting pore. The relative amount and size of the leachable substance gives rise to the degree of porosity. Also, as described herein, porosity can be achieved using sintering and / or by controlling the treatment cycle of glass gels to control the pores and interpores of the material.

[0093] The bioactive glass can be prepared in several ways, to provide melt-derived bioactive glass, sol-gel derived bioactive glass, and sintered bioactive glass particles. The sintered particles may be in solgel derived, or pre-reacted melt derived form. Sol-gel derived glass is generally prepared by synthesizing an inorganic network by mixing metal alkoxides in solution, followed by hydrolysis, gelation, and low temperature (around 200 to 900 °C) firing to produce a glass. Sol-gel derived glasses produced this way are known to have an initial high specific surface area compared with either melt-derived glass or porous melt-derived glass. The surface area of the sol-gel derived glasses is at least about 50 m2 / g. Melt derived bioactive glass is generally prepared by mixing grains of oxides or carbonates, melting and homogenizing the mixtures at high temperatures, typically between about 1250 and 1400 °C. The molten bioactive glass can be fritted and milled to produce a small particulate material, e.g., a powder.

[0094] The bioactive glass is preferably melt-derived. In each preparation, it is preferred to use reagent grade bioactive glass, especially since the glass is used to prepare materials which ultimately may be administered to a patient. Melt derived bioactive glass

[0095] Melt derived bioactive glass can be prepared, for example, by preparing an admixture of the individual metal oxides and other components used to prepare the glass composition, blending the admixture, melting the admixture, and cooling the mixture. The melting temperature is determined in large part by the glass composition, and ranges, for example, from about 900 to1500 °C, preferably between about 1250 and 1450 °C. The melt is preferably mixed, for example, by oxygen bubbling, to ensure a thorough homogenization of the individual components.

[0096] The melt can be cooled, for example, by adding the molten admixture to a suitable liquid, such as deionized water, to produce a glass frit. Porosity can be introduced by grinding the glass into a powder, admixing the powder with a foaming agent, and hot pressing the mixture under vacuum and elevated temperature. The particle size of the bioactive glass powder is between about 2 and 70 pm, the vacuum is preferably less than 50 MPa, and the hot pressing is preferably performed at a temperature above 400 °C, preferably between about 400 and 500 °C. Suitable foaming agents include compounds which evolve carbon dioxide and / or water at elevated temperatures, for example, metal hydroxides, metal carbonates, and peroxides, such as hydrogen peroxide. Preferred metal carbonates are sodium bicarbonate, sodium carbonate and calcium carbonate. The foaming agents are preferably added in a range of between about 1 to 5, more preferably 2 to 3 wt.% of the bioactive glass powder. The preparation of melt-derived porous glass is described, for example, in U.S. Patent No. 5,648,301 the contents of which are hereby incorporated by reference.

[0097] Sintered bioactive glass particles

[0098] Bioactive glass can be sintered using known methodology. In one embodiment, an aqueous slurry of the bioactive glass powder and a foaming agent with a suitable binder, such as polyvinyl alcohol, is formed. The slurry is then poured into a mold, allowed to dry, and sintered at high temperatures. This temperature may range, depending on the glass composition and foaming agent used, from about 500 to 1000 °C, more preferably from about 600 to 800 °C.

[0099] Spun Fibers of Sol-gel derived bioactive glass

[0100] It is known in the art to control the heat treatment cycle of bioactive glass gels to control the pores and interpores of the material to create a porous glass material. However, since a pore diameter larger than 0.1 microns is difficult to achieve using this method, the sintering and foaming processes described herein are generally more preferred.

[0101] Leaching of the porous material To aid in preparing bioactive glass with high porosity, the bioactive glass can include a material which can be preferably leached out of the bioactive glass, and, in doing so, provide it with high porosity. For example, minute particles of a material capable of being dissolved in a suitable solvent, acid, or base can be mixed with or melted into the bioactive glass, and subsequently leached out. The resulting voids have roughly the same size as the particle that was leached out. In the case of a material which is part of a melt-derived glass composition, the size of the pores and degree of porosity depends on the amount of added material relative to the amount of glass. For example, if the leached material constituted about 80 percent of the glass, then the glass would be approximately 80 percent greater than porous when the material was leached out. When leaching the glass composition, care should be taken not to leach out those components which add to the bioactivity of the glass, i.e., the calcium and phosphorus oxides.

[0102] In the method according to the present invention at least one additive can be optionally present.

[0103] In one embodiment the at least one additive is a growth factor, for example a fibroblast growth factor, epidermal growth factor, nerve growth factor or connective tissue growth factor or a recombinant human bone morphogenesis protein.

[0104] In one embodiment the at least one additive is selected from thrombin, fibrinogen, chitosan, silicic acid precursors, heparin, heparin derived oligosaccharides, hyaluronic acid, alginate and glycosaminoglycans.

[0105] In one embodiment of the method, the the lyophilization step is performed at -40 to -60 °C; and / or the obtained hydrogel is cooled to -20 to -80 °C before the lyophilization step is performed.

[0106] In one embodiment the aqueous solution is shaken for 1 to 24 hours before the lyophilization step is performed.

[0107] By performing the method according to the invention, a sponge is obtained.

[0108] In one embodiment the sponge has a water uptake capacity of 500 to 3000 %, based on the total dry weight of the sponge.

[0109] In one embodiment the sponge has a total fluid uptake of 0.5 - 2 g.

[0110] In one embodiment the sponge has a swelling of 5 to 45 % after fluid uptake.

[0111] In one embodiment the sponge has a Young’s modulus of 1 to 10 kPa in wet form.

[0112] In one embodiment the sponge has a pore size of 15 to 300 pm. The above-mentioned characteristics of the sponge are preferably determined as described in the example section. By shredding, milling or fragmentation of the sponge according to the present invention particles can preferably be obtained with an average particle size in the range of 30 to 300 pm. The particle size can preferably be determined by light scattering or sieving.

[0113] The sponge of the present invention can be used for bone regeneration, cartilage repair, skin repair or as dental membrane.

[0114] Protein sequences SEQ ID NO:1 Streptomyces pyogenes Collagen-like protein (CLP), full length protein

[0115] SEQ ID NO:2 Streptomyces pyogenes CLP, truncation 3

[0116] SEQ ID NO:3 Streptomyces pyogenes CLP, truncation 5

[0117] SEQ ID NO:4 Streptomyces pyogenes CLP, no V-domain

[0118] Examples

[0119] In the examples, collagen-like protein was obtained according to the following method and is referred to as “rCol” as well.

[0120] Production of collagen-like protein

[0121] The bacterial collagen-like protein was produced in different host cells by fermentation.

[0122] To produce Scl2 from Streptomyces pyogenes in Pichia pastoris, the sequence of the collagen domain of the gene scl2, encoding for a collagen-like protein, has been codon optimized using different algorithms, and cloned in a secretion vector for Pichia pastoris and transformed in Pichia pastoris following standard protocol and subsequent application of a standard expression protocol in fed-batch mode, protein corresponding to Scl2p was detected in the supernatant of cell culture. (Damasceno, L.M., Huang, CJ. & Batt, C.A. Protein secretion in Pichia pastoris and advances in protein production. Appl Microbiol Biotechnol 93, 31-39 (2012)).

[0123] Upon fermentation, supernatant has been separated from biomass via centrifugation (12000g, 5 mins at room temperature).

[0124] The protein could be produced under similar conditions using either E. coli or C. glutamicum. In case of a production in yeast or C. glutamicum, the CL single strand is secreted by the cell. No cell lysis is needed as an initial purification step in this approach. In case of a production in E. coli a cell lysis is mandatory to remove the product from the cell.

[0125] The full-length collagen-like protein, a truncated variant (truncation 3) and the no-V-domain variant (based on the gene sc / 2 from Streptomyces pyogenes) were also expressed in Brevibacillus choshinensis. Therefore, the corresponding DNA sequences were cloned into a suitable secretion vector for B. choshinensis. Transformation of B. choshinensis with the new constructed plasmids was done according to Mizukami et al. 2010 (Curr Pharm Biotechnol 2010, 13: 151 -258).

[0126] The B. choshinensis strains were analyzed for their ability to produce the different collagen proteins in batch cultivations at 33°C and pH 7 using the DASGIP® parallel bioreactor system from Eppendorf (Hamburg, Germany). The fermentation was performed using 1 L reactors. The production medium (TM medium, Biomed Res Int 2017, 2017: 5479762) contained 10 g / L glucose. Upon fermentation, supernatant has been separated from biomass by centrifugation and was used for SDS PAGE analysis. For all three variants, collagen-like protein was produced.

[0127] The full-length collagen-like protein and the no-V-domain variant (based on the gene scl2 from Streptomyces pyogenes) were also expressed in Corynebacterium glutamicum. Therefore, the corresponding DNA sequences were cloned together with an upstream located signal peptide for protein secretion into a shuttle vector for C. glutamicum (Biotechnology Techniques 1999, 13: 437- 441.). The C. glutamicum strain ATCC 13032 was transformed with the new constructed plasmids by means of electroporation as described by Ruan et al. (Biotechnology Letters 2015, 37: 2445- 2452).

[0128] The C. glutamicum strains were analysed for their ability to produce the different collagen proteins in fed-batch cultivations at 30°C and pH 7 using the DASGIP® parallel bioreactor system from Eppendorf (Hamburg, Germany). The fermentation was performed using 1 L reactors. The production medium contained 20 g / L glucose in the batch phase and the fed-batch phase was run with a glucose feed of 4 g / L*h. Upon fermentation, supernatant has been separated from biomass by centrifugation and was used for HPLC analysis. For both variants, collagen protein was produced. For the truncated variant of the collagen-like protein, product titer was higher as for the full-length variant.

[0129] After cell separation (via centrifugation) and folding of the bacterial collagen-like protein (via cooling of the concentrate) the bacterial collagen-like protein was purified using precipitation with 2-Propanol at 15 v%. After precipitation of the Scl2 protein a centrifugation was performed. The pellet was dissolved in water, the triple helical Scl2 protein was unfolded at 40°C and filtered through a 100 kD membrane. This step serves to remove large sized impurities. The collected permeate was then concentrated in the consecutive 10 kDa filtration. The retentate was washed to remove small sized impurities.

[0130] By that means a triple helical Scl2 protein purity >75 w% was achieved.

[0131] In the comparative examples animal derived collagen, i.e., rat tail collagen, commercially available from Sigma Aldrich under product number C7661 is used and is referred to as “rtCol” as well.

[0132] Example 1: Composite made of the bacterial collagen-like protein and bioactive glass

[0133] The composite was made of calcium sodium phosphosilicate powder (material number MD01 , Schott, Product No 1137402, Lot SM699), composed of very small particles with a D50 value of 3.9 pm and a D99 value of 14.5 pm, and consisting of 24.5+3 wt% CaO, 24,5+3 wt% Na2O, 6+2 wt% P2O5, 45±3 wt% SiO2. This bioactive glass powder was first pre-mixed in millipore H2O, thereafter, transferred to the equal weight of the bacterial collagen-like protein powder. For comparison, sponges made from 25 mg / ml, 50 mg / ml and 75 mg / ml bacterial collagen-like protein solutions, all supplemented with equal amount of bioactive glass were exemplarily manufactured. All components were shortly vortexed and orbitally shaken overnight at 450 rpm. After overnight dissolution time, sedimented glass particles were homogenously dispersed by resuspending. Thereafter, the dispersion was transferred to a standard polystyrene plastic mold (24-well plate) and frozen to -80 °C for 4 hours. A volume of 1 ml dispersion was frozen to manufacture sponges being applied for most of the described analysis. Only exception was the samples applied for structure analysis, here the samples were made of 0.4 ml dispersion solution. After pre-freezing, a Christ LSC Plus freeze dryer was pre-cooled to -45 °C before placing the samples within the drying chamber. The pre-frozen samples were dried in vacuo at 0.07 mbar, and the temperature was stepwise increased to 20 °C (for details see Table 1). After the lyophilization step, the dried spongy scaffold was stabilized by dehydrothermal treatment in vacuo at 50 mbar and heated at 140°C for 24 hours.

[0134] Table 1: Process applied for the lyophilization of composite sponges with collagen-like protein and bioactive glass.

[0135] Example 2: Test of transferability of the established process to animal-derived collagen

[0136] Initially bioactive glass (5 mg or 45 mg) was pre-mixed in 1 ml of 0.1 % acetic acid solution, usually applied to dissolve animal-derived collagens. The prepared dispersion was transferred to 5 mg lyophilized rat tail collagen (Sigma, C7661-100mg). All components were vortexed and orbitally shaken at 450 rpm overnight. After overnight incubation at room temperature the rat tail collagen was not dissolved. Photos were taken and pH was measured of formulations with bioactive glass and of the pure solvent, 0.1 % acetic acid. pH of the collagen solution increased rapidly to a value of 10-11 leading to a sedimentation of the collagen material. This proof-of-concept study clearly shows that the formation of a homogeneous solution was not feasible with animal-derived collagen, impeding further processing to sponges.

[0137] Absorption, and swelling of sponges after fluid contact

[0138] Sponges were submerged in 5 ml phosphate-buffered saline, equal to approximately fivefold the volume of the samples, for 24 hours at room temperature to analyze fluid absorption and swelling characteristics. Additionally, photos were taken before and after hydration to observe the integrity and swelling behavior. The fluid absorption and absolute fluid uptake were calculated by weight measurement of the dried and hydrated samples, as summarized by following equations: fluid uptake [^] — '^'hydrated — ^dry The effect of swelling was calculated by the diameter change (Ad) after hydration of the sponges, as expressed by the equation below: . ioo

[0139] Mechanical compression of sponges

[0140] After hydration for 24 hours in phosphate-buffered saline, sponges were compressed with a Brookfield CT3 texture analyzer (maximum load of 4500 g). This mechanical testing device was equipped with a probe (11.3 mm diameter, 1 cm2surface) slightly smaller than the samples’ diameter (16 mm). The compression was performed with a measurement velocity of 0.5 mm / s. The applied weight and the respective distance were captured during the compression process. The recorded raw data was used to calculate the stress and the strain, finally plotting both values as a function of each other. Young’s moduli were derived as the slope in the linear elastic range (approximately from 10 to 20 % strain).

[0141] Pore size of sponges

[0142] Sponges were frozen in liquid nitrogen and cut with a blade to create a brittle fracture cross-sectional surface. Sections were fixed with a sticky pad on a SEM sample stub. The sample surface was sputtered with gold-palladium, thereby increasing electrical conductivity. With a scanning electron microscope (SEM, high vacuum, 10kV, SE-mode), secondary electron pictures were captured in 250- fold and 2500-fold magnification. The pore size of several cells was measured (n = 20) with an image analysis software

[0143] Cell seeding with Cal-72 cells

[0144] Cal-72 bone cells were seeded on top of the sponges to show the biocompatibility and the cell interaction of developed composites. Before cell seeding, sponges were transferred to a 24-well plate, and sterilized with UV light for 30 minutes, turning the upside down after 15 minutes. For optimal seeding efficiency, sponges were pre-incubated with 1 ml DMEM medium (supplemented with 10 % fetal calf serum, 2 mM glutamine, 1x insulin-transferrin-sodium selenite and 30 pg / ml gentamicin) overnight at 37 °C. Cal-72 bone cells (5 * 105per sponge) were applied drop-by-drop in total volume of 0.1 ml culture medium on top of the sponges. After a first adherence time of 2 hours incubating the construct at 37 °C, the sponges were transferred to a 12-well plate and further 0.9 ml cell culture medium was added to the well. The constructs were cultured for further 7 days, and medium was exchanged every second to third day. After this culture period, cell viability was stained with MTT dye (1 mg / ml) in phosphate-buffered saline for 4 hours at 37 °C. After incubation, photos of the stained composite were taken with a top view and with a cross-section view.

Claims

Claims1. Method of preparing a bioactive glass containing sponge based on collagen-like protein comprising or consisting of the steps: i) providing an aqueous solution comprising at least one collagen-like protein, at least one bioactive glass and optionally at least one additive; ii) performing a lyophilization step to obtain a sponge; iii) thereafter performing a dehydrothermal treatment to the sponge obtained in step ii); and iv) optionally sterilizing the sponge obtained after step iii).

2. Method according to claim 1, wherein the bioactive glass is i) a calcium sodium phosphosilicate; and / or ii) present in 5 to 75 mg / ml in the aqueous solution.

3. Method according to claim 1 or 2, wherein the at least one collagen-like protein is i) a bacterial collagen-like protein; and / or ii) present in the aqueous solution with a concentration range from 2.5 to 200 mg / ml.

4. Method according to any of the preceding claims, wherein the at least one additive is i) selected from a growth factor, thrombin, fibrinogen, chitosan, silicic acid precursors, heparin, heparin derived oligosaccharides, hyaluronic acid, and glycosaminoglycans and / or ii) present in 0.001 to 10 mg / ml in the aqueous solution.

5. Method according to any of the preceding claims, wherein the lyophilization step i) is performed at -40 to -60 °C; or ii) is performed at -40 to -60 °C and the obtained dispersion is cooled to -20 to -80 °C; before the lyophilization step is performed.

6. Method according to any of the preceding claims, wherein the aqueous solution is shaken for 1 to 24 hours before the lyophilization step is performed.

7. Method according to any of the preceding claims, wherein the dehydrothermal treatment is performed at 110 to 160 °C and under 40 to 60 mbar vacuum and 3 to 48 h.

8. Sponge obtained by the method according to any of claims 1 to 7.

9. Sponge according to claim 8, having i) a water uptake capacity of 500 to 3000 %, based on the total dry weight of the sponge; and / orii) a fluid up take of 0.5 to 2 g; and / or iii) a swelling of 5 to 45 %; and / or iv) a Young’s modulus of 1 to 10 kPa in wet form; and / or v) a pore size of 15 to 300 pm.

10. Particles obtained by shredding, milling or fragmentation of the sponge according to claim 8 or 9.

11. Use of the sponge according to claim 8 or 9 or the particles according to claim 10 for bone regeneration, cartilage repair, skin repair or as dental membrane.

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