IN SITU MINERALIZATION OF 3D PRINTED METASTABLE CALCIUM SPECIES

Abstract

The present invention refers to a biomimetic minerizable 3D-printing ink, a method for the production of such a biomimetic minerizable 3D-printing ink, a method for the production of a biomineralized 3D-printed article, a biomineralized 3D-printed article as well as the use of a crystallization trigger which is an oligopeptide selected from the group comprising an oligopeptide of the HABP family and an oligopeptide of the P11-family for 3D printing.

Claims

1. A biomimetic minerizable 3D-printing ink comprising a) a calcium cation-based compound being metastable calcium carbonate or metastable calcium phosphate, b) a carrier material, and c) a crystallization trigger.

2. The biomimetic minerizable 3D-printing ink according to claim 1, wherein the calcium cation-based compound has a crystallinity of less than 50 wt.-%, preferably of less than 40 wt.-%, more preferably of less than 30 wt.-% and most preferably of less than 20 wt.-%, based on the total weight of the calcium cation-based compound.

3. The biomimetic minerizable 3D-printing ink according to claim 1, wherein the calcium cation-based compound has a weight median particle size d.sub.50 as determined by dynamic light scattering in the range from 1 to 500 nm, preferably from 50 to 400 nm and most preferably from 100 to 350 nm.

4. The biomimetic minerizable 3D-printing ink according to claim 1, wherein the carrier material is a material suitable to form a hydrogel, preferably the carrier material is selected from the group comprising gelatin, methylcellulose, alginate, agarose, fibrin, hyaluronic acid, proteins such as gelatin, nidogen, collagen and heparan sulfate proteoglycans and mixtures thereof, K-carrageenan, poly(ethylene glycol) (PEG), polycaprolactone (PCL), poloxamer, peptide and mixtures thereof.

5. The biomimetic minerizable 3D-printing ink according to claim 1, wherein the crystallization trigger is a peptide, an oligopeptide or a protein, preferably the crystallization trigger is an oligopeptide, more preferably the crystallization trigger is an oligopeptide comprising 11 amino acid residues and comprising a hydrophobic aromatic core.

6. The biomimetic minerizable 3D-printing ink according to claim 1, wherein the crystallization trigger is an oligopeptide selected from the group comprising an oligopeptide of the HABP family, preferably HABP1 and HABP2, and an oligopeptide of the P11-family, preferably selected from the group consisting of P11-4, P11-8, P11-9, P11-12, P11-13, P11-14, P11-15, P11-16, P11-17, P11-18, P11-19, P11-20, P11-24, P11-25, P11-26, P11-27, P11-28, P11-29, P11-30, P11-31, P11-32 and mixtures, and most preferably P11-4, optionally the oligopeptide of the P11-family is associated with a negatively charged polysaccharide or a positively charged polysaccharide.

7. The biomimetic minerizable 3D-printing ink according to ay ene of claim 1, wherein the crystallization trigger is an oligopeptide in which the amino acids at positions 4 and 8 are phenylalanine (F) and the amino acid at position 6 is tryptophan (W) and/or the amino acid residues at both positions 5 and 7 of the peptide are ornithine (O) or glutamic acid (E).

8. The biomimetic minerizable 3D-printing ink according to claim 1, wherein the ink comprises a) the calcium cation-based compound in an amount ranging from 2 to 30 wt.-%, preferably from 5 to 25 wt.-%, and most preferably from 10 to 25 wt.-%, based on the total weight of the ink, and b) the carrier material in an amount ranging from 1 to 10 wt.-%, preferably from 1 to 8 wt.-%, and most preferably from 1 to 5 wt.-%, based on the total weight of the ink, and c) the crystallization trigger in an amount ranging from 0.05 to 3 wt.-%, preferably from 0.05 to 2.8 wt.-%, and most preferably from 0.1 to 2.5 wt.-%, based on the total weight of the ink, and d) a buffer solution in an amount ranging from 57 to 96.95 wt.-%, preferably from 64.2 to 93.95 wt.-%, and most preferably from 67.5 to 98.9 wt.-%, based on the total weight of the ink.

9. The biomimetic minerizable 3D-printing ink according to claim 1, wherein the calcium cation-based compound being metastable calcium carbonate or metastable calcium phosphate comprises a stabilizer,

10. The biomimetic minerizable 3D-printing ink according to claim 9, wherein the stabilizer is selected from the group comprising magnesium chloride, polyaspartic acid, glutamic acid, polyacrylic acid, phosphates such as L-O-phosphoserine, sodium dihydrogen phosphate and disodium hydrogen phosphate, saccharides, EDTMP, xanthan, polysorbate, citric acid, ehylenediamine, extracts from biogenic samples, double-hydrophilic block copolymers and mixtures thereof.

11. A method for the production of a biomimetic minerizable 3D-printing ink as defined in claim 1, the method comprising the steps of a) providing a calcium cation-based compound being metastable calcium carbonate or metastable calcium phosphate, b) providing a carrier material, c) providing a crystallization trigger, and d) mixing the calcium cation-based compound being metastable calcium carbonate or metastable calcium phosphate of step a), the carrier material of step b) and the crystallization trigger of step c).

12. A method for the production of a biomineralized 3D-printed article, the method comprising the steps of a) providing a biomimetic minerizable 3D-printing ink as defined in claim 1, b) printing the biomimetic minerizable 3D-printing ink into a predetermined form by using a 3D-printer, and c) hardening the biomimetic minerizable 3D-printing ink at a temperature ranging from 10 to 50 C., preferably from 15 to 45 C., for obtaining the biomineralized 3D-printed article.

13. The method according to claim 12, wherein the hardening in step c) is carried out at a) a temperature ranging from 15 to 40 C. and most preferably at a temperature ranging from 20 to 40 C., and/or b) a hardening time ranging from 2 hours to 21 days, preferably from 12 hours to 19 days and most preferably from 24 hours to 18 days, and/or c) a CO.sub.2 content in the atmosphere from 3 to 6 vol.-%, preferably 4 to 6 vol-% and most preferably from 4.5 to 5.5 vol-%, and/or d) a humidity of more than 75 vol. %, preferably in the range from 80 to 100 vol. % and most preferably in the range from 85 to 99.5 vol.-%.

14. The method according to claim 12, wherein the method comprises a further step d) of drying the biomineralized 3D-printed article obtained in step c), preferably at a temperature of at least 50 C., more preferably at a temperature ranging from 60 to 100 C. and most preferably from 65 to 90 C., and/or a drying time ranging from 5 hours to 36 hours, preferably from 5 hours to 32 hours and most preferably from 6 hours to 26 hours.

15. A biomineralized 3D-printed article according to claim 12.

16. The biomineralized 3D-printed article according to claim 15, wherein the article is a dental reconstruction material, ceramic substitute, mollusk shell substitute, nacre substitute, dentin substitute, tooth substitute or bone substitute.

17. The method of claim 11, wherein the crystallization trigger is an oligopeptide selected from the group comprising an oligopeptide of the HABP family and an oligopeptide of the P11-family.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0228] FIG. 1 shows SEM pictures of the prepared ACP (left) and ACP after printing (right)

[0229] FIG. 2 shows a SEM picture of the prepared ACC

[0230] FIG. 3 shows a hardness comparison for the prepared ACP inks

[0231] FIG. 4 shows the Vickers hardness for example 2

[0232] FIG. 5 shows the Vickers hardness for example 3

[0233] FIG. 6 shows the Vickers hardness for example 4

[0234] FIG. 7 shows a hardness comparison for the prepared ACC inks

[0235] FIG. 8 shows a XRD comparison between ACP before and after 3D printing example 4

[0236] FIG. 9 shows a FTIR comparison between ACP before and after 3D printeding example 4

EXAMPLES

Materials Used

[0237] CaCl.sub.2*2H.sub.2O99% of Sigma Aldrich [0238] Na.sub.2CO.sub.399.5% of Sigma Aldrich [0239] L-O-phosphoserine 98% of TCI [0240] Ca(NO.sub.3).sub.2*4H.sub.2O99% of Sigma Aldrich [0241] (NH.sub.4).sub.2HPO.sub.498% of Sigma Aldrich [0242] NH.sub.4OH-solution (28.0-30.0% NH.sub.3 basis) of Sigma Aldrich [0243] Whatman Cellulose Nitrate Membrane Filters, 9 cm, 0.8 m [0244] Fann filter, special hardened filter paper, 9 cm, 2-5 m [0245] Amorphous calcium phosphate ACP and amorphous calcium carbonate ACC: Omya AG [0246] Methyl cellulose (MC) of Sigma Aldrich [0247] NaOH of Sigma Aldrich [0248] Na.sub.2HPO.sub.4 of Sigma Aldrich [0249] Citric Acid of Sigma Aldrich [0250] 1PBS (pH=7.4) of Sigma Aldrich [0251] Peptide P11-4 (sequence: CH3CO-QQRFEWEFEQQ-NH2) of Credentis AG

Preparation of ACP and ACC

[0252] ACP preparation: Precipitation of calcium phosphate salts in aqueous solution was performed in that an aqueous solution of diammonium phosphate was stirred, while adding in one step the calcium nitrate tetrahydrate aqueous solution. In this step, a precipitation was observed. The ACP was recovered by filtration and cleaning steps followed by freeze drying.

[0253] The details of the solution preparation and of the synthesis are set out in the following tables 1 and 2.

TABLE-US-00001 TABLE 1 solution preparation for ACP Phosphate Calcium Rinse Solution solution Solution 0.15M 0.35M 0.14 wt % (NH.sub.4).sub.2HPO.sub.4 [g] 15.4 Ca(NO.sub.3).sub.2 *4H.sub.2O [g] 27.5 NH.sub.4OH (28-30 wt %) [ml] 23.6 24.8 2.43 Dest. water [ml] 746.4 308.2 1797.57 Total volume [ml] 777 333 1800

TABLE-US-00002 TABLE 2 Synthesis of ACP Phosphate Calcium solution solution 0.15M 0.35M Mixing 65 ml 27.5 ml Calcium solution was added to 1200 rpm, phosphate solution with stirring, 30 sec precipitation took place. Filtration fann filter, Part Nr. 206051 Precipitation was separated 9 cm, pore size 2-5 m from liquid phase by approx. 15 min filtration. Rinsing Rinse solution filter cake was rinsed during 150 ml filtration Freeze ~48 h 1 mbar, 54 C. drying Manually ground and sieved Grinding in a 100 m sieve and sieving d.sub.50 ~236 nm BET SSA 64.69 m.sup.2/g

[0254] The final products were stored in a desiccator until further uses.

[0255] FIG. 1 shows SEM pictures of the prepared ACP (left) and ACP after printing (right).

[0256] ACC preparation: Precipitation of calcium carbonate salts in aqueous solution was performed in that an aqueous solution of sodium carbonate was stirred while adding in one step the calcium chloride aqueous solution. In this step, a precipitation was observed. The ACC was recovered by filtration and cleaning steps followed by freeze drying.

[0257] The details of the solution preparation and of the synthesis are set out in the following tables 3 and 4.

TABLE-US-00003 TABLE 3 solution preparation for ACC Carbonate solution 0.25M Calcium solution with stabilizer 0.08M 0.25M NaCO.sub.3 [g] 13.248 CaCl.sub.2 [g] 18.376 L-O-Phosphoserine [g] 7.403 MilliQ [ml] 500 500 Total Volume 500 500 pH 9.38 6.95

TABLE-US-00004 TABLE 4 Synthesis of ACC Carbonate Calcium Solution 0.25M solution (stabilizer) 0.25M Mixing 40 ml 40 ml Calcium solution (5 C.) was 1200 rpm, added to carbonate solution 30 sec (5 C.) with stirring, precipitation took place. Filtration Cellulose Nitrate Membrane Precipitation was separated 9 cm, pore size 0.8 m from liquid phase by approx. 15 min filtration. Rinsing Cold miliQ (5 C.) filter cake was rinsed during 50 ml filtration Freeze ~48 h 1 mbar, 54 C. drying Manually ground and sieved Grinding in a 100 m sieve and sieving d.sub.50 ~332 nm BET SSA 28.03 m.sup.2/g

[0258] The final product was stored in a desiccator until further uses.

[0259] FIG. 2 shows a SEM picture of the prepared ACC.

Preparation of Solutions

[0260] Stock Solution MC 15 cp (Methyl Cellulose) light (7.4% w/w) [0261] Dissolve 8 g MC into 100 g PBS (Phosphate Buffered Saline) and let swell overnight at 4 C. [0262] P11-4 Peptide (1%, 3% w/w) (for 3 ml)adapted accordingly for different ml [0263] Dissolve peptide P11-4 in a solution of 0.04 M Na.sub.2HPO.sub.4, 1 M NaOH and 0.02 M citric acid.

Characterization Methods

TGA

[0264] Thermal gravimetrical analysis was performed on a TGA 4000 from Perkin Elmer. The TGA program was hold for 1.0 min at 30.00 C. and heat from 30.00 C. to 900.00 C. at 10.00 C./min.

BET

[0265] Powder specific surface area was measured with Micromeritics ASAP 2460. The degassing of the sample was done under vacuum. Samples were measured according to norm ISO 9277.

Particle Size Distribution

[0266] Particle size distribution was performed by dynamic light scattering by using a Malvern Zetasizer Nano ZS. For the sample preparation, a dispersed solution containing 0.1 wt % of the particles in an aqueous solution and 0.0021 wt % of polyacrylate dispersant was prepared. The suspension was high shear mixed for 3 minutes and then ultrasonicated for 30 minute. The sample was measured.

Vickers Experiment

[0267] The measurement was performed with Struers DURAMIN-40 M1 (https://www.struers.com/en/Products/Hardness-testing/Hardness-testing-equipment/Duramin-40).

[0268] To obtain a flat surface for indentation, the samples were grinded (1000 grade paper), polished (cotton cloth) and cleaned with a fiberless paper towel by hand. Surface was flushed with N.sub.2 after the analysis. The indentation position was chosen by optical inspection and navigation with micrometer stage. Then, the load appropriate for HV0.01 was applied. The tip dwelled in the force load position for 10s before being retracted. The size of the indent was measured optically, followed by an automatic evaluation of the Vickers hardness.

[0269] The instruments works according to the following norms: ASTM E384, ISO 6507 and JIS Z 2244.

XRD

[0270] About 0.16 cm.sup.3 of sample was loaded into a PMMA specimen holder, offering a flat, circular (d=20 mm) surface for XRD analysis. The sample was then analyzed obeying Bragg's law, using a Bruker D8 Advance ECO powder diffractometer with a 1 kW X-ray tube operating 40 kV and 25 mA, a - (Bragg-Brentano) goniometer, and a LYNXEYE XE-T detector, scanning from 3 to 70 2 at 0.02 2 steps for 0.5 s per step. Nickel-filtered Cu K.sub. radiation (=1.54060-10.sup.10 m) was employed in all experiments. During measurement, the sample was rotated at 15 rpm to maximize random distribution of analyzed crystal surfaces. The resulting powder diffraction pattern was interpreted qualitatively with respect to mineral content using the Bruker DIFFRACsuite software package EVA in comparison to the ICDD PDF library of reference patterns.

FTIR (Perkin-Elmer)

[0271] FTIR-spectra of the sample in attenuated total reflection (ATR) for type verification. The FTIR spectra were collected using a single bounce ATR unit (Gladi-ATR). The spectral data were slightly smoothened (value 20), baseline corrected and normalized to 1.5 A (absorbance which corresponds to 3.16% transmission)

Field Emission Scanning Electron Microscope (FESEM)

[0272] Field emission scanning electron microscope (Zeiss Sigma VP) was used for investigations. Two different detectors were involved: [0273] 1. Images from secondary electron detector (SE) show particle shape of the sample [0274] 2. InLens secondary electron detector (InLens) was used to show fine structures.

EXAMPLES

[0275] Before the ink preparation, the gel liquid materials MC and peptides were prepared as follow: [0276] 1. 8 g of MC was dissolved in 100 g PBS (Phosphate Buffered Saline) and left to swell overnight at 4 C. [0277] 2. 90.63 mg of peptide P11-4 was dissolved in 100.6 l of 1M NaOH/1.365 ml of 0.04 Na.sub.2HPO.sub.4. As final step for self-assembling, 1.5 ml of 0.02 M of citric acid was added.

[0278] For the ACP ink preparation, all the materials were mixed: 1.sup.st the gel liquid materials (MC and P11-4) were mixed, followed by the addition of the mineral (ACP).

[0279] 3D-printed article samples were prepared by using the 3D printer RegenHU 3Discovery via direct extrusion trough a metallic syringe needle (ID 0.3 mm) or conical PLA needle (ID 0.4 mm). The ink was filled in a 3 ml plastic syringe, tip was mounted. The ink was extruded by applying a pneumatic pressure until a flow rate of 1-2 mm/s was measured. Then, the 3D-printed article samples were printed in the form of squares of 11 cm line by line and layer by layer (each layer 3 mm high in total 3 layers) through computer controlled motion of the motorized 3D stages.

[0280] The 3D-printed article samples were stored in an incubator at 37 C., 99 vol-% humidity and 5 vol-% CO.sub.2 for defined intervals of time (0.04, 017, 1, 2, 6, 9, 16 days) for hardening.

[0281] The 3D-printed article samples were further dried for 24 hours at 70 C. and stored in a desiccator for further characterization in Vickers, XRD and FTIR.

[0282] The 3D-printed article samples prepared are set out in the following table 5.

TABLE-US-00005 TABLE 5 3D-printed article samples prepared based on ACP Example 1 Example 2 Example 3 Example 4 ACP (% w/w) 20 20 20 20 MC (% w/w) 5.9 4.5 1.5 1.5 P11-4 (% w/w) 0.2 0.6 1.8 Buffer aqueous 74.1 75.3 77.9 76.7 solution (% w/w) Printability +++ ++ +++ +++

[0283] For the ACC ink preparation all the materials were mixed: 1.sup.st the gel liquid materials (MC and P11-4) were mixed, followed by the addition of the mineral (ACC).

[0284] 3D-printed article samples were prepared by using the 3D printer RegenHU 3Discovery via direct extrusion trough a metallic syringe needle (ID 0.3 mm) or conical PLA needle (ID 0.4 mm). The ink was filled in a 3 ml plastic syringe, tip was mounted. The ink was extruded by applying a pneumatic pressure until a flow rate of 1-2 mm/s was measured. Then, the 3D-printed article samples were printed in the form of squares of 11 cm line by line and layer by layer (each layer 3 mm high in total 3 layers) through computer controlled motion of the motorized 3D stages.

[0285] The 3D-printed article samples were stored in an incubator at 37 C., 99 vol-% humidity and 5 vol-% CO.sub.2 for 16 days for hardening.

[0286] The 3D-printed article samples were further dried for 24 hours at 70 C. and stored in a desiccator for further characterization in Vickers, XRD and FTIR.

[0287] The 3D-printed article samples prepared are set out in the following table 6.

TABLE-US-00006 TABLE 6 3D-printed article samples prepared based on ACC Example 5 Example 6 ACC (% w/w) 20 20 MC (% w/w) 5.9 4.5 P11-4 (% w/w) 0.2 Buffer aqueous 74.1 75.3 solution (% w/w) Printability +++ ++

[0288] The use of a crystallization trigger such as a peptide is of advantage to increase the hardness of the constructs once being 3D printed. This was evidenced by the hardness measurements and comparison of the formulations that contain higher concentration of the crystallization trigger as can be seen in FIGS. 3, 4, 5, and 6, (examples 2, 3, 4 and 6). These samples show even higher hardness than the control (teeth). The time also plays a role on the increase of the hardness, this is possible to see on the higher crystallization trigger content formulations (example 3 and 4). This suggested again that the crystallization trigger is strongly involved in the hardening of the construct. It was also elucidated that the amorphous phase transformed towards the crystal phases, shown by XRD and FTIR (FIG. 8 and FIG. 9). In particular, the peak broadening shown for the ACP in FIG. 8 confirms that metastable calcium phosphate, and especially amorphous calcium phosphate, is present having a crystallinity of less than 20 wt.-%, based on the total weight of the calcium phosphate.