COMPOSITION BASED ON RECOMBINANT BIOPOLYMERS AND USES OF SAME AS BIO-INK

Abstract

The present invention refers to compositions comprising recombinant biopolymers made of combinations of monomers of the type “Elastin-like recombinamers” (ELR), monomers comprising the “silk” sequence and/or monomers comprising the HLF sequence that belongs to a natural class of proteins named zippers. Said compositions are useful as bio-ink for 3D printing. Furthermore, the present invention also refers to methods for obtaining the composition of the invention, as well as the 3D biomaterial and to the different uses of the composition and the obtained biomaterial.

Claims

1. A composition comprising a biopolymer comprising monomers B, C, X and Y, or at least two biopolymers comprising monomers B, C and X and monomers B, C and Y, respectively, wherein B comprises repeats of the pentapeptide VPGXG (SEQ ID NO: 7), C is an amino acid sequence comprising SEQ ID NO: 3, X is an amino acid sequence comprising SEQ ID NO: 4, and Y comprises 1 to 15 repeats of SEQ ID NO: 8.

2. The composition according to claim 1, wherein the biopolymer further comprises monomer D, said monomer D being a cell-binding amino acid sequence.

3. The composition according to claim 2, wherein monomer D comprises a sequence selected from the list consisting of: RGD (SEQ ID NO: 9), LDT (SEQ ID NO: 27), SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 28 or SEQ ID NO: 29.

4. The composition according to claim 2, wherein monomer D comprises SEQ ID NO: 6.

5. The composition according to claim 1 wherein the biopolymer has structure (I):
[(B.sub.b−C.sub.c)−Z.sub.z].sub.n−D.sub.d wherein B comprises repeats of the pentapeptide VPGXG (SEQ ID NO: 7), C is an amino acid sequence comprising SEQ ID NO: 3 and D is a cell-binding amino acid sequence, Z is selected from monomers X and/or Y, wherein X is an amino acid sequence comprising SEQ ID NO: 4 and Y comprises 1 to 15 repeats of SEQ ID NO: 8, b has values of between 5 and 15, c has values of between 50 and 70; z has values of between 1 and 5, n has values of between 1 and 5, d has values of between 0 and 3, and it is characterized in that it comprises a first biopolymer of structure (I), wherein Z is SEQ ID NO: 4, and a second biopolymer of structure (I), wherein Z is SEQ ID NO: 5.

6. The composition according to claim 5, wherein the first biopolymer is found in the composition at a concentration of between 40-60% by weight and the second biopolymer is found in the composition at a concentration of between 60-40% by weight.

7. The composition according to claim 1, wherein the biopolymer has structure (II):
Z1.sub.z−[(B.sub.b−C.sub.c)−Z2.sub.z].sub.n−D.sub.d, wherein B comprises repeats of the pentapeptide VPGXG (SEQ ID NO: 7), C is an amino acid sequence comprising SEQ ID NO: 3 and D is a cell-binding amino acid sequence, Z1 is an amino acid sequence comprising SEQ ID NO: 4 or SEQ ID NO: 5 and Z2 is an amino acid sequence comprising SEQ ID NO: 5 or SEQ ID NO: 4, respectively; b has values of between 5 and 15, c has values of between 50 and 70, z has values of between 1 and 5, n has values of between 1 and 5 and d has values of between 0 and 3.

8. The composition according to claim 5, wherein b has a value of 10, c has a value of 60, z has a value of 1, n has a value of 2 and d has a value of 0 or 1.

9. The composition according to claim 2, wherein the biopolymer is selected from SEQ ID NO: 16 or from the combination of biopolymers of SEQ ID NO: 14 and SEQ ID NO: 15.

10. The composition according to claim 1, further comprising cells, bioactive molecules, active ingredients, or combinations thereof.

11. The composition according to claim 1, characterized in that it is in the form of a hydrogel.

12. (canceled)

13. (canceled)

14. (canceled)

15. (canceled)

16. A bio-ink comprising the composition according to claim 1.

17. A 3D or 2D biomaterial comprising the composition according to claim 1.

18. A drug comprising the composition according to claim 1.

19. A method for tissue regeneration comprising administering the drug according to claim 18 to a subject in need thereof.

20. A bio-ink comprising a composition comprising a biopolymer comprising monomers B, C and at least monomer X or Y, wherein: B comprises repeats of the pentapeptide VPGXG (SEQ ID NO: 7), C is an amino acid sequence comprising SEQ ID NO: 3, X is an amino acid sequence comprising SEQ ID NO: 4, and comprises 1 to 15 repeats of SEQ ID NO: 8.

21. (canceled)

22. (canceled)

23. (canceled)

24. (canceled)

25. (canceled)

26. (canceled)

27. (canceled)

28. The composition according to claim 1, wherein B is an amino acid sequence comprising SEQ ID NO: 2.

29. The composition according to claim 1, wherein Y is an amino acid sequence comprising SEQ ID NO: 5.

30. The bio-ink according to claim 20, wherein B is an amino acid sequence comprising SEQ ID NO: 2.

31. The bio-ink according to claim 20, wherein Y is an amino acid sequence comprising SEQ ID NO: 5.

32. The composition according to claim 7, wherein b has a value of 10, c has a value of 60, z has a value of 1, n has a value of 2 and d has a value of 0 or 1.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0099] FIG. 1 shows acrylamide gel electrophoresis of biopolymer 1 with the molecular weight marker in the lane on the left and biopolymer 1 in the lane on the right. The molecular weights are indicated in kilodaltons (kDa).

[0100] FIG. 2 shows a mass spectroscopy (MALDI-ToF, or “Matrix-assisted laser desorption/ionization-time of flight”) analysis of biopolymer 1 which shows the value of its experimental molecular mass of 92897 Da, wherein the theoretical molecular mass is 93175 Da and the difference between both can be attributed to the measurement error. The monodisperse nature of the molecule is also observed, with only one narrow peak being seen.

[0101] FIG. 3 shows an infrared spectroscopy (FTIR-ATR, or “Fourier Transform Infrared-Attenuated Total Reflectance”) analysis of biopolymer 1 which shows the characteristic signals of the amide groups (˜1700 cm-1) present in the designed protein biopolymers.

[0102] FIG. 4 shows a nuclear magnetic resonance (NMR) analysis of biopolymer 1 in which the signal of the hydrogens belonging to the amine group NH (7.5-8.5 ppm), to the methyl group CH.sub.3 (0.5-1.0 ppm) and to the methylene group CH (1.0-2.3; 3.5-4.5 ppm) is observed.

[0103] FIG. 5 shows acrylamide gel electrophoresis of biopolymer 2 with the molecular weight marker in the lane on the right and biopolymer 2 in the lane on the left. The molecular weights are indicated in kilodaltons (kDa).

[0104] FIG. 6 shows a MALDI-TOF analysis of biopolymer 2 which shows the value of its experimental molecular mass of 101664 Da, wherein the theoretical molecular mass is 101696 Da and the difference between both can be attributed to the measurement error.

[0105] FIG. 7 shows a FTIR-ATR analysis of biopolymer 2 which shows the characteristic signals of the amide groups (˜1700 cm-1) present in the designed protein polymers.

[0106] FIG. 8 shows a nuclear magnetic resonance (NMR) analysis of biopolymer 2 in which the signal of the hydrogens belonging to the amine group NH (7.5-8.5 ppm), to the methyl group CH.sub.3 (0.5-1.0 ppm) and to the methylene group CH (1.0-2.3; 3.5-4.5 ppm) is observed.

[0107] FIG. 9 shows acrylamide gel electrophoresis of biopolymer 3 with the molecular weight marker in the lane on the left and biopolymer 3 in the lane on the right. The molecular weights are indicated in kilodaltons (kDa).

[0108] FIG. 10 shows a MALDI-TOF analysis of biopolymer 3 which shows the value of its experimental molecular mass of 103793 Da, wherein the theoretical molecular mass is 104119 Da and the difference between both can be attributed to the measurement error.

[0109] FIG. 11 shows a FTIR-ATR analysis of biopolymer 3 which shows the characteristic signals of the amide groups (˜1700 cm-1) present in the designed protein polymers.

[0110] FIG. 12 shows a nuclear magnetic resonance (NMR) analysis of biopolymer 3 in which the signal of the hydrogens belonging to the amine group NH (7.5-8.5 ppm), to the methyl group CH.sub.3 (0.5-1.0 ppm) and to the methylene group CH (1.0-2.3; 3.5-4.5 ppm) is observed.

[0111] FIG. 13 shows acrylamide gel electrophoresis of biopolymer 4 with the molecular weight marker in the lane on the right and biopolymer 4 in the lane on the left. The molecular weights are indicated in kilodaltons (kDa).

[0112] FIG. 14 shows a MALDI-TOF analysis of biopolymer 4 which shows the value of its experimental molecular mass of 122882 Da, wherein the theoretical molecular mass is 123345 Da and the difference between both can be attributed to the measurement error.

[0113] FIG. 15 shows a FTIR-ATR analysis of biopolymer 4 which shows the characteristic signals of the amide groups (˜1700 cm-1) present in the designed protein polymers.

[0114] FIG. 16 shows a nuclear magnetic resonance (NMR) analysis of biopolymer 4 in which the signal of the hydrogens belonging to the amine group NH (7.5-8.5 ppm), to the methyl group CH.sub.3 (0.5-1.0 ppm) and to the methylene group CH (1.0-2.3; 3.5-4.5 ppm) is observed.

[0115] FIG. 17 shows acrylamide gel electrophoresis of biopolymer 5 with the molecular weight marker in the lane on the right and biopolymer 5 in the lane on the left. The molecular weights are indicated in kilodaltons (kDa).

[0116] FIG. 18 shows a MALDI-TOF analysis of biopolymer 5 which shows the value of its experimental molecular mass of 120611 Da, wherein the theoretical molecular mass is 120921 Da and the difference between both can be attributed to the measurement error.

[0117] FIG. 19 shows a FTIR-ATR analysis of biopolymer 5 which shows the characteristic signals of the amide groups (˜1700 cm-1) present in the designed protein polymers.

[0118] FIG. 20 shows a nuclear magnetic resonance (NMR) analysis of biopolymer 5 in which the signal of the hydrogens belonging to the amine group NH (7.5-8.5 ppm), to the methyl group CH.sub.3 (0.5-1.0 ppm) and to the methylene group CH (1.0-2.3; 3.5-4.5 ppm) is observed.

[0119] FIG. 21 shows acrylamide gel electrophoresis of biopolymer 6 with the molecular weight marker in the lane on the right and biopolymer 6 in the lane on the left. The molecular weights are indicated in kilodaltons (kDa).

[0120] FIG. 22 shows a MALDI-TOF analysis of biopolymer 6 which shows the value of its experimental molecular mass of 125857 Da, wherein the theoretical molecular mass is 126393 Da and the difference between both can be attributed to the measurement error.

[0121] FIG. 23 shows a FTIR-ATR analysis of biopolymer 6 which shows the characteristic signals of the amide groups (˜1700 cm-1) present in the designed protein polymers.

[0122] FIG. 24 shows a nuclear magnetic resonance (NMR) analysis of biopolymer 6 in which the signal of the hydrogens belonging to the amine group NH (7.5-8.5 ppm), to the methyl group CH.sub.3 (0.5-1.0 ppm) and to the methylene group CH (1.0-2.3; 3.5-4.5 ppm) is observed.

[0123] FIG. 25 shows photographs of the biomaterial printed with the composition comprising different concentrations (300, 250, 200, 180, 150 and 120 mg/ml) of pre-cured biopolymer 5 (SEQ ID NO: 15) shown in column A, and of biopolymer 4 (SEQ ID NO: 14) shown in column B, using PBS1× as solvent.

[0124] FIG. 26 shows photographs of different biomaterials printed with the different compositions of the invention at a concentration of 250 mg/ml using PBS1× as solvent, wherein the printability (Column A) and fibrillar observation (Column B) of said biomaterials are clearly shown. BP: Biopolymer. The percentage of biopolymer combinations refers to the percentage expressed by weight.

[0125] FIG. 27 shows viscosity (expressed in Pascals per second, Pa.$) of the bio-inks formed by different biopolymers of the invention subjected to an increasing shear velocity (1/s).

[0126] FIG. 28 shows an evaluation of the variation in viscosity in different bio-inks of the invention subjected to a high shear velocity for a short time interval. Step 1: Shear velocity of 5 s.sup.−1. Step 2: Shear velocity of 1000 s.sup.−1. Step 3: Shear velocity of 5 s.sup.−1.

[0127] FIG. 29 shows the effect of temperature on the viscosity of different analyzed bio-inks of the invention.

[0128] FIG. 30 shows photographs of different structures printed with the bio-ink comprising biopolymer 4 (SEQ ID NO: 14) using PBS1× as solvent, where stability of the printed structures over 3 days is shown.

[0129] FIG. 31 shows photographs of different structures printed with the bio-ink comprising pre-cured biopolymer 5 (SEQ ID NO: 15) using PBS1× as solvent, where stability of the printed structures over 2 days is shown.

[0130] FIG. 32 shows photographs of different structures printed with the bio-ink comprising the combination of biopolymers, 60% by weight of biopolymer 4 (SEQ ID NO: 14) and 40% by weight of pre-cured biopolymer 5 (SEQ ID NO: 15) using PBS1× as solvent, where stability of the printed structures over 40 days is shown.

[0131] FIG. 33 shows photographs of different structures printed with the bio-ink comprising biopolymer 6 (SEQ ID NO: 16) using PBS1× as solvent, where stability of the printed structures over 40 days is shown.

[0132] FIG. 34 shows a graph showing an analysis of early cell adhesion at times of 30 min, 2 hours and 4 hours, of the mixtures of biopolymer 4 (60% by weight) of SEQ ID NO: 14 and pre-cured biopolymer 5 (40% by weight) of SEQ ID NO: 15, comprising the RGD adhesion sequence (white blocks), and of the mixture of biopolymer 3 (60% by weight) of SEQ ID NO: 13 and biopolymer 2 (40% by weight) of SEQ ID NO: 12, not comprising cell adhesion sequence (black blocks).

[0133] FIG. 35 shows an analysis of cell proliferation over 21 days on printed surfaces based on mixtures of biopolymer 4 (60% by weight) of SEQ ID NO: 14 and pre-cured biopolymer 5 (40% by weight) of SEQ ID NO: 15, comprising the RGD adhesion sequence (white blocks), and of the mixture of biopolymer 3 (60% by weight) of SEQ ID NO: 13 and biopolymer 2 (40% by weight) of SEQ ID NO: 12, not comprising cell adhesion sequence (black blocks).

[0134] FIG. 36 shows a microscopic photograph of a surface printed with the bio-ink comprising the combination of biopolymer 4 (60% by weight) of SEQ ID NO: 14 and pre-cured biopolymer 5 (40% by weight) of SEQ ID NO: 15 using PBS1× as solvent, on which HFF-1 cells have been seeded and cultured for 7 days.

[0135] FIG. 37 shows microscopic photographs of a surface printed with the bio-ink comprising the combination of biopolymer 4 (60% by weight) of SEQ ID NO: 14 and pre-cured biopolymer 5 (40% by weight) of SEQ ID NO: 15 using PBS1× as solvent, on which HFF-1 cells have been seeded and cultured for 7 days. Different focal planes are shown to corroborate the three-dimensionality of the system.

[0136] FIG. 38 shows the viability of human fibroblasts HFF-1 printed together with biopolymer 6 over 21 days. Viability of the printed gratings is compared together with the control (non-printed deposited material).

[0137] FIG. 39 shows microscopic photographs of a surface printed with biopolymer 6 mixed with human fibroblasts HFF-1 over 21 days. The scale corresponds to 500 μm.

EXAMPLES

[0138] The invention is illustrated below by means of assays conducted by the inventors, describing the synthesis of the composition of the invention, as well as its features. The examples are provided for the purpose of understanding the description and are not intended to limit the present invention.

Example 1. Obtaining and Characterization of the Recombinant Protein Biopolymers Forming the Composition of the Invention

[0139] The design and obtaining of synthetic nucleotide sequences coding for the amino acid sequences of the different biopolymers used, including the biopolymers comprising the composition of the invention, was performed as described in WO/2010/092224. Likewise, the expression, purification and characterization of the biopolymers was carried out as described in WO/2010/092224.

[0140] Briefly, the ELRs are designed and produced by means of recombinant DNA technologies. Once the nucleotide sequence coding for the desired protein has been introduced in bacterial strain Escherichia coli, said strain is subjected to culturing in a fermenter, which allows absolute control of the production conditions. When the stationary phase in the bacterial culture growth curve is achieved, the desired ELR is extracted by means of ultrasonic lysis of the bacterial wall. Purification of the biopolymer will be carried out using its inverse temperature transition property, performing bacterial debris heating and cooling cycles until obtaining the pure polymer.

[0141] After a salt elimination process through dialysis, all the biopolymers used are lyophilized, showing a whitish and cottony appearance, and are set aside until they are used in this state at −20° C.

[0142] To characterize the obtained biopolymers, the following techniques are used: [0143] Acrylamide gel electrophoresis (PAGE) in the presence of SDS which allows the molecular weight of the biopolymer to be approximately estimated, in addition to the purity thereof to be verified. [0144] MALDI-TOF mass spectrometry in a Q-Star spectrometer to precisely obtain the molecular weight of the polymer. [0145] Proton nuclear magnetic resonance (H1-NMR) spectrum in a Bruker ARX300 spectrometer. [0146] Infrared (FT-IR) spectrum using a Cary 50 spectrophotometer. [0147] HPLC chromatography with UV detection using a WATERS 600 HPLC gradient system with a WATERS 2487 detector, which allows the amino acid composition to be determined. [0148] Differential scanning calorimetry (DSC) of aqueous solutions of the material with a concentration of 50 mg/ml in Mettler Toledo 822e DSC equipment, to obtain the inverse transition temperature of the polymer.

[0149] To demonstrate the effectiveness of the composition of the invention, specifically for use as a bio-ink, the following biopolymers were designed (Table 2), to subsequently determine which are the best compositions for use as bio-inks.

TABLE-US-00002 TABLE 2 Biopolymers, structure and amino acid and nucleotide sequences: Amino acid Nucleotide Name Structure sequence sequence Biopolymer 1 {(B10-C60)}2-D SEQ ID NO: SEQ ID NO: (93175 Da) 11 21 Biopolymer 2 {(B10-C60)-Y}2 SEQ ID NO: SEQ ID NO: (101696 Da) 12 22 Biopolymer 3 {(B10-C60)-X}2 SEQ ID NO: SEQ ID NO: (104119 Da) 13 23 Biopolymer 4 {(B10-C60)-X}2-D SEQ ID NO: SEQ ID NO: (123345 Da) 14 24 Biopolymer 5 {(B10-C60)-Y}2-D SEQ ID NO: SEQ ID NO: (120921 Da) 15 25 Biopolymer 6 X-{(B10-C60)-Y}2-D SEQ ID NO: SEQ ID NO: (126393 Da) 16 26

Biopolymer 1 (1107 Amino Acids)

[0150] Structure: (A)−{(B.sub.10−C.sub.60)}.sub.2−D.

TABLE-US-00003 Amino acid sequence: SEQ ID NO: 11: MESLLP-{[VPGVG).sub.2-(VPGEG)- (VPGVG).sub.2].sub.10[VGIPG].sub.60}.sub.2-([VPGIG].sub.5AVTGRGDSPASS).sub.6-V

[0151] Coded by nucleotide sequence SEQ ID NO: 21.

[0152] The theoretical amino acid composition and the amino acid composition obtained with HPLC with UV (ultraviolet light) detection are presented in Table 3.

TABLE-US-00004 TABLE 3 Analysis of the amino acid composition of biopolymer 1. Glu Gly Ile Leu Pro Ser Val Amino Theoretical 21 440 120 2 221 1 301 acid Experimental 22.01 432.83 119.34 1.91 224.02 1.09 304.80 analysis

[0153] The production yield was 227.65 mg/l.

[0154] The theoretical molecular weight for biopolymer 1 is 93175 Da and it was experimentally estimated by polyacrylamide gel electrophoresis (FIG. 1) and by MALDI-TOF mass spectrometry, resulting in 92897 Da. HPLC, infrared (IR) and nuclear magnetic resonance (NMR) spectra obtained for biopolymer 1 are shown in FIGS. 2, 3 and 4, respectively.

[0155] The transition temperature obtained by means of DSC in MQ at pH 7.8 was 19.10° C., while in 1×PBS at pH 7.65 it was 14.66° C.

Biopolymer 2 (1233 Amino Acids)

[0156] Structure: (A)−{(B.sub.10−C.sub.60)−Y}.sub.2

TABLE-US-00005 Amino acid sequence SEQ ID NO: 12: MESLLP-{([(VPGVG).sub.2-(VPGEG)- (VPGVG).sub.2].sub.10[VGIPG].sub.60)[V(GAGAGS).sub.5G].sub.2}.sub.2

[0157] Coded by nucleotide sequence SEQ ID NO: 22.

[0158] The theoretical amino acid composition and the amino acid composition obtained with HPLC with UV (ultraviolet light) detection are presented in Table 4.

TABLE-US-00006 TABLE 4 Analysis of the amino acid composition of biopolymer 2. Ala Glu Gly Ile Leu Amino Theo- 40 21 504 120 2 acid retical analysis Experi- 33.17 24.57 501.30 122.87 2.46 mental Met Pro Ser Val Amino Theo- 1 221 21 305 acid retical analysis Experi- 1.72 232.22 17.20 294.39 mental

[0159] The production yield was 178.9 mg/l.

[0160] The theoretical molecular weight for polymer 2 is 101696 Da and it was experimentally estimated by polyacrylamide gel electrophoresis (FIG. 5) and by MALDI-TOF mass spectrometry (FIG. 6) resulting in 101664 Da. IR and NMR spectra obtained for biopolymer 2 are shown in FIGS. 7 and 8, respectively.

[0161] The transition temperature obtained by means of DSC in MQ at pH 6.14 was 20.08° C., while in 1×PBS at pH 6.40 it was 16.92° C.

Biopolymer 3 (1213 Amino Acids)

[0162] Structure: (A)−{(B.sub.10−C.sub.60)−X}.sub.2

TABLE-US-00007 Amino acid sequence SEQ ID NO: 13: MESLLP-{[VPGVG)2-(VPGEG)-(VPGVG).sub.2].sub.10[VGIPG].sub.60- [VGGGGGKENQIAIRASFLEKENSALRQEVADLRKELGKCKN ILAKYEAGGGGG]}.sub.2

[0163] Coded by nucleotide sequence SEQ ID NO: 23.

[0164] The theoretical amino acid composition and the amino acid composition obtained with HPLC with UV (ultraviolet light) detection are presented in Table 5.

TABLE-US-00008 TABLE 5 Analysis of the amino acid composition of biopolymer 3. Ala Arg Asn Asp Cys Glu Gln Gly Ile Amino Theoretical 12 6 6 2 2 33 4 462 126 acid Experimental 12.17 5.35 6.17 1.94 0.97 33.53 4.51 468.61 136.08 analysis Leu Lys Met Phe Pro Ser Tyr Val Amino Theoretical 12 12 1 2 221 5 2 305 acid Experimental 11.44 10.20 1.72 2.70 238.81 5.68 2.72 273.06 analysis

[0165] The production yield was 517.22 mg/l.

[0166] The theoretical molecular weight for polymer F is 104119 Da and it was experimentally estimated by polyacrylamide gel electrophoresis (FIG. 9) and by MALDI-TOF mass spectrometry (FIG. 10) resulting in 103,793 Da. IR and NMR spectra obtained for biopolymer 3 are shown in FIGS. 11 and 12, respectively.

[0167] The transition temperature obtained by means of DSC in MQ at pH 7.5 was 15.30° C., while in 1×PBS at pH 7.5 it was 14.18° C.

Biopolymer 4 (1435 Amino Acids)

[0168] Structure: (A)−{(B.sub.10−C.sub.60)−X}.sub.2−D

TABLE-US-00009 Amino acid sequence SEQ ID NO: 14: MESLLP-{[VPGVG).sub.2-(VPGEG)- (VPGVG).sub.2].sub.10[VGIPG].sub.60- [VGGGGGKENQIAIRASFLEKENSALRQ EVADLRKELGKCKNILAKYEAGGGG G]}.sub.2-([VPGIG].sub.5AVTGRGDSPASS).sub.6-V

[0169] Coded by nucleotide sequence SEQ ID NO: 24.

[0170] The theoretical amino acid composition and the amino acid composition obtained with HPLC with UV (ultraviolet light) detection are presented in Table 6.

TABLE-US-00010 TABLE 6 Analysis of the amino acid composition of biopolymer 4. Ala Arg Asp + Asn Cys Glu + Gln Gly Ile Leu Amino Theoretical 24 12 8 + 6 2 33 + 34 534 156 12 acid Experimental 18.34 6.50 11.72 1.97 37.83 563.8 148.32 11.84 analysis Lys Met Phe Pro Ser Thr Tyr Val Amino Theoretical 12 1 2 257 23 6 2 341 acid Experimental 9.75 1.39 1.16 283.18 12.65 3.83 2.67 350.61 analysis

[0171] The production yield was 239.81 mg/l.

[0172] The theoretical molecular weight for biopolymer 4 is 123345 Da and it was experimentally estimated by polyacrylamide gel electrophoresis (FIG. 13) and by MALDI-TOF mass spectrometry (FIG. 14) resulting in 122882 Da. IR and NMR spectra obtained for biopolymer 4 are shown in FIGS. 15 and 16, respectively.

[0173] The transition temperature obtained by means of DSC in MQ at pH 6.48 was 17.58° C., while in 1×PBS at pH 6.02 it was 14.92° C.

Biopolymer 5 (1455 Amino Acids)

[0174] Structure: (A)−{(B.sub.10−C.sub.60)−Y}.sub.2−D

TABLE-US-00011 Amino acid sequence SEQ ID NO: 15: MESLLP-{([(VPGVG).sub.2-(VPGEG)- (VPGVG).sub.2].sub.10[VGIPG].sub.60)[V(GAGAGS).sub.5G].sub.2}.sub.2- ([VPGIG].sub.5AVTGRGDSPASS).sub.6-V

[0175] Coded by nucleotide sequence SEQ ID NO: 25.

[0176] The theoretical amino acid composition and the amino acid composition obtained with HPLC with UV (ultraviolet light) detection are presented in Table 7.

TABLE-US-00012 TABLE 7 Analysis of the amino acid composition of biopolymer 5. Ala Arg Asp Glu Gly Ile Amino Theoretical 52 6 6 21 574 150 acid Experimental 50.94 4.31 5.39 29 581.86 149.41 analysis Leu Met Pro Ser Thr Val Amino Theoretical 2 1 257 39 6 341 acid Experimental 0 1.37 255.77 31.08 4.33 335.86 analysis

[0177] The production yield was 203.07 mg/l.

[0178] The theoretical molecular weight for biopolymer 3 is 120921 Da and it was experimentally estimated by polyacrylamide gel electrophoresis (FIG. 17) and by MALDI-TOF mass spectrometry (FIG. 18) resulting in 120611 Da. IR and NMR spectra obtained for biopolymer 2 are shown in FIGS. 19 and 20, respectively.

[0179] The transition temperature obtained by means of DSC in MQ at pH 6.59 was 20.84° C., while in 1×PBS at pH 7.24 it was 17.26° C.

Biopolymer 6 (1508 Amino Acids)

[0180] Structure: (A)−X−{(B.sub.10−C.sub.60)−Y}.sub.2−D

TABLE-US-00013 Amino acid sequence SEQ ID NO: 16: MESLLP- [VGGGGGKENQIAIRASFLEKENSALRQEVADLRKE LGKCKNILAKYEAGGGG G]-{([(VPGVG).sub.2-(VPGEG)- (VPGVG).sub.2].sub.10[VGIPG].sub.60)-[V(GAGAGS).sub.5G].sub.2}.sub.2- ([VPGIG].sub.5AVTGRGDSPASS).sub.6-V

[0181] Coded by nucleotide sequence SEQ ID NO: 26.

[0182] The theoretical amino acid composition and the amino acid composition obtained with HPLC with UV (ultraviolet light) detection are presented in Table 8.

TABLE-US-00014 TABLE 8 Analysis of the amino acid composition of biopolymer 6. Ala Arg Asn + Asp Cys Glu + Gln Gly Ile Leu Amino Theoretical 58 9 3 + 7 1 27 + 2 585 153 7 acid Experimental 53.78 7.72 11.15 0.32 38.85 583.93 147.01 8.90 analysis Lys Met Phe Pro Ser Thr Tyr Val Amino Theoretical 6 1 1 257 41 6 6 343 acid Experimental 12.37 0.98 1.13 260.95 35.50 4.54 13.91 323.98 analysis

[0183] The production yield was 116 mg/l.

[0184] The theoretical molecular weight for biopolymer 6 is 126393 Da and it was experimentally estimated by polyacrylamide gel electrophoresis (FIG. 21) and by MALDI-TOF mass spectrometry (FIG. 22) resulting in 125857 Da. IR and NMR spectra obtained for biopolymer 6 are shown in FIGS. 23 and 24, respectively.

[0185] The transition temperature obtained by means of DSC in MQ at pH 7.50 was 20.42° C., while in 1×PBS at pH 7.50 it was 17.41° C.

Example 2. Determination of the Composition of the Bio-Ink of the Invention which Allows Optimal Printing

[0186] 3D printing with the different biopolymers described in Table 2, or with mixtures thereof, are performed taking into account the inverse transition temperature of each of them. Said transition temperature together with the specific properties of the compositions of the biopolymers of the invention cause the potential bio-inks to gel by means of a simple change in temperature.

[0187] The experimental system used comprises a REGEMAT 3D printer on which there has been installed a head connected to a cooling bath, which allows the injection temperature to be kept at 4° C. Moreover, the printer has a heating bed which is kept at 30° C. during the printing process.

[0188] In the case of biopolymer 1 (SEQ ID NO: 11), gelling is due to the hydrophobic intermolecular forces present between its blocks C (hydrophobic) and B (hydrophilic). Block D, specifically comprising the peptide RGD additionally introduced in its sequence, does not affect gel formation, but rather is introduced to provide the biopolymer with biofunctionality. This biopolymer 1 will be used as a negative control of bioprinting, given that it does not contain any of monomer X, monomer Y or both.

[0189] The rest of biopolymers 2 (SEQ ID NO: 12), 3 (SEQ ID NO: 13), 4 (SEQ ID NO: 14), 5 (SEQ ID NO: 15) and 6 (SEQ ID NO: 16), containing base monomers C (hydrophobic) and B (hydrophilic), in addition to other monomers X and/or Y, also show these hydrophobic interactions. They all further contain block D comprising, in the examples shown, specifically peptide RGD to provide the biopolymer with biofunctionality, allowing cell adhesion to be induced.

[0190] Biopolymers 4 (SEQ ID NO: 14) and 3 (SEQ ID NO: 13) comprise the zipper sequence (SEQ ID NO: 4), which allows the formation of alpha helices through the interaction of electrostatic forces between charged amino acids, contributing to the stability of the polymer. Biopolymers 5 (SEQ ID NO: 15) and 2 (SEQ ID NO: 12) show the same hydrophobic interactions, but in this case stabilized as a result of the formation of beta sheets originating from the silk sequence (SEQ ID NO: 8), by means of the formation of hydrogen bonds between amido and carboxyl groups present in their amino acids.

[0191] For the particular case of biopolymer 5 (SEQ ID NO: 15), a comparison between its gelling when said biopolymer has been subjected to a pre-curing treatment (referred to hereinafter as pre-cured biopolymer 5) or when it has not been subjected to said treatment (which will continue to be called biopolymer 5) has been carried out. The pre-curing treatment is performed due to the variability of biopolymer 5 in terms of its structure at a molecular level, where it may present a different degree of formation of beta sheets which affect its mechanical characteristics. During the production and purification of biopolymer 5, the formation of beta sheets occurs through hydrogen bonds. Said cross-linking is not homogeneous among the different batches of biopolymer, generating batches having a different initial cross-linking. Breaking the hydrogen bonds with the pre-curing treatment ensures that the initial state of beta sheet formation is the same for all the batches. To carry out the pre-curing treatment, first the biopolymer is homogenized by breaking its intermolecular forces using formic acid, which allows starting from a state with an absence of beta sheets. After this state, the biopolymer is subjected to curing at 37° C. for 24 hours, favoring the formation of beta sheets. The same initial state is thereby ensured in all the batches of this polymer and the process starts from a pre-gelled state that is foreseeably more suitable for printing.

[0192] Both biopolymer 1 (SEQ ID NO: 11) mixed together with biopolymer 4 (SEQ ID NO: 14), and biopolymer 1 (SEQ ID NO: 11) mixed with pre-cured biopolymer 5 (SEQ ID NO: 15) also serve as a negative control for printing. Their printing demonstrates that optimal printing is obtained only if the mixture comprises both reinforcement sequences (FIG. 26).

[0193] Moreover, for the purpose of confirming whether the mixtures of polymer 4 and pre-cured polymer 5 behave the same way when monomers X and Y are located in the same biopolymer, biopolymer 6 (SEQ ID NO: 6) was synthesized. This biopolymer 6 presents both the initial hydrophobic interactions and the electrostatic and hydrogen bond interactions originating from the Zipper and Silk sequences.

[0194] Different printing operations were carried out with compositions comprising biopolymers 1, 4, 5, pre-cured biopolymer 5 and biopolymer 6, alone or combined. To this end, by means of the REGEMAT 3D printer software, a grating is designed, measuring 10×10 mm, with a height of 1.30 mm (corresponding to 6 layers of height), and a porosity of 1.5 mm arranged at an angle of 90°. The different compositions of the biopolymers of the invention will be injected with a 0.25 mm nozzle and 0.06 mm/s flow. The flow is adapted if needed in each case to enable making lines of similar width which allow the structures to be better compared to one another.

[0195] The optimal printing concentration of pre-cured biopolymer 5 dissolved at different concentrations of 1×PBS (120, 150, 180, 200, 250 and 300 mg/ml) is estimated to be 250 mg/ml, as observed in FIG. 25, given that said concentration is the concentration at which the viscosity of the solution is suitable for the “injection” process by means of the 3D printer. Therefore, the concentration of 250 mg/ml is selected for the comparison of the various printing operations. For the case of biopolymer 4, as also observed in FIG. 25, the suitable concentration for the injection process in the 3D printer is 250 mg/ml.

[0196] Printing the designed gratings allows semi-quantification of the printability of the various biopolymers of the invention to be performed by measuring printability, which gives an idea as to the similarity the printed structure has with respect to designed structure. In this case, square pores are designed and printed, therefore a parameter (Pr) is established for measuring the similarity of the printing operations with respect to these squares.

[0197] The bio-ink demonstrating good printability must be deposited through the extrusion of filaments having a constant morphology which allow being deposited in height, without being fused to one another. If the bio-ink demonstrates less printability, this does not occur and the filaments tend to collapse and fuse to one another, forming porosities that tend to be circular. Circularity is therefore defined as:

[00001]$C=\frac{4\pi A}{L^2}$

[0198] Wherein L defines the perimeter and A the area. The closer the obtained value is to 1, the greater the circularity will be, with 1 being a perfect circle.

[0199] In the case of square shapes, circularity will be π/4, and parameter Pr based on the square shape can be defined as:

[00002]$\mathrm{Pr}=\frac{\pi }{4}.Math.\frac{1}{C}=\frac{L^2}{16A}$

[0200] For ideal printability, the interconnected pores will be square and the Pr value will be 1.

[0201] The determination of parameter Pr is performed by taking photographs with a LEICA DMS 1000 digital microscope, and the optical images taken are analyzed through Image J software (n=5) (FIG. 26).

[0202] The results of said parameter for the assayed samples are summarized in Table 9.

TABLE-US-00015 TABLE 9 Measurement of printability (Pr) obtained in each bio-ink printed at a final concentration of 250 mg/ml. BIO-INKS (250 mg/ml) Pr VALUE biopolymer 1 — biopolymer 4 0.94 ± 0.03 biopolymer 5 — pre-cured biopolymer 5 0.90 ± 0.09 biopolymer 6 0.97 ± 0.10 biopolymer 1 (40%) + biopolymer 4 (60%) 0.77 ± 0.04 biopolymer 1 (60%) + biopolymer 5 (40%) 0.73 ± 0.10 biopolymer 4 (60%) + biopolymer 5 (40%) 0.86 ± 0.04 biopolymer 4 (20%) + pre-cured biopolymer 5 (80%) 0.65 ± 0.32 biopolymer 4 (60%) + pre-cured biopolymer 5 (40%) 0.96 ± 0.04 biopolymer 4 (80%) − pre-cured biopolymer 5 (20%) 0.93 ± 0.02

[0203] Another parameter, in this case qualitative, for determining printability of bio-inks is fibrillar observation, in other words, if the deposition of the polymers is deposited layer-by-layer and the deposited fibers are observed, the structure will tend to collapse less than if these fibers fuse to one another, causing lower shape fidelity.

[0204] In FIG. 26, the photographs of the printing that was performed are observed. After measuring parameter Pr, it is observed that after a Pr value of 0.90, the printing operations are performed in a controlled manner, allowing the deposition of fibers which do not mix with one another when they are deposited, maintaining their fibrillar structure in height.

[0205] As observed in FIG. 26, biopolymer 1 does not gel after printing, therefore it does not maintain its structure. This behavior corresponds with what is expected, given that this polymer does not contain monomers X and/or Y, which allow for stability of the gel formed.

[0206] Unlike what occurs with biopolymer 1, when biopolymers have monomers X and/or Y, as in the case of biopolymers 4, 5 and pre-cured biopolymer 5, printing is performed with same. The biopolymer 5 allows for printing thereof, but the printed structure rapidly disaggregates such that it does not allow its printability to be measured. In contrast, pre-cured polymer 5 shows over-gelling, which is observed in the non-linear deposition of the fibers, despite its high printing fidelity. Therefore, the fact that the sample gels due to a change in temperature is not a sufficient condition as would have been expected for achieving a good bio-ink for this system, and thus obtaining printed surfaces showing a structure which truly resembles the designed structure. The phenomenon that accompanies gelling and the modifications in the mechanical properties of the material before and after gelling are determining factors and may be inadequate, without being able to predict which material is suitable as a bio-ink.

[0207] Lastly, biopolymer 4 shows reliable structures with Pr values of 0.94 (FIG. 26).

[0208] Given that pre-cured polymer 5 shows a higher printability parameter than polymer 5, for demonstrating the synergistic effect of monomers X and Y, pre-cured polymer 5 is selected as a carrier of monomer Y, such that the following mixtures are printed: biopolymer 4 (80% by weight)+pre-cured biopolymer 5 (20% by weight); biopolymer 4 (60% by weight)+pre-cured biopolymer 5 (40% by weight) and biopolymer 4 (20% by weight)+pre-cured biopolymer 5 (80% by weight). The printing of the proportions of biopolymer 4 (60% by weight)+pre-cured biopolymer 5 (40% by weight) show the highest Pr value obtained in the printed mixtures, therefore this mixture can be considered optimal for 3D printing. The lowest value is obtained when the proportion of pre-cured biopolymer 5 is higher. Furthermore, printing of the mixture of biopolymer 4 (60% by weight)+biopolymer 5 (40% by weight) allows observing that when the printing mixture for polymers of this type is optimal, if biopolymer 5 is not subjected to pre-curing, printability decreases, making it necessary to use pre-cured biopolymer 5 if better structure fidelity is to be obtained.

[0209] To confirm the synergy between monomers X and Y, the mixtures comprising biopolymer 5 (40% by weight)+biopolymer 1 (60% by weight) and the mixture of biopolymer 4 (60% by weight)+biopolymer 1 (40% by weight) are also printed. Said mixtures show printing with worse printability, confirming that to obtain good printability, the mixture of biopolymers comprising monomers X and Y is necessary.

[0210] Lastly, the printing of biopolymer 6 shows very good shape fidelity in which the deposition of fibers is observed, such that they do not end up being completely superimposed. The highest printability value, corresponding to 0.97, is obtained for this polymer. Although the Pr value obtained for this polymer is not very different from that of the mixture comprising biopolymer 4 (60% by weight)+pre-cured biopolymer 5 (40% by weight), greater deposition ease is observed in printing, with better control over the deposited fibers.

Example 3. Mechanical Properties of the Compositions of the Invention

[0211] A rheological analysis was carried out to analyze the mechanical properties of the compositions of the invention for use as bio-inks. Those compositions which demonstrated having greater printability are selected, corresponding with the compositions comprising biopolymer 4, biopolymers 6, pre-cured biopolymer 5 and the mixture of biopolymers 4 (60% by weight)+pre-cured biopolymer 5 (40% by weight).

[0212] The rheological study is carried out with a TA Instruments AR 200 EX rheometer, equipped with a peltier plate, and a geometry of 40 mm in diameter. All the analyzed compositions are kept at 4° C. during the assays.

[0213] The first characterization is based on the study of the variation in viscosity experienced by the compositions of the invention for use as bio-inks when subjected to an increasing shear velocity (FIG. 27). Both biopolymer 4 and the mixture comprising biopolymer 4 (60% by weight)+pre-cured biopolymer 5 (40% by weight) do not show a decrease in viscosity when shear velocity is increased, therefore behaving like Newtonian fluids showing relatively low viscosities (about 1 Pa.$). The fact that they behave like low-viscosity Newtonian fluids allows controlled depositions to be performed with lower shear stresses, thus protecting the cells which are embedded in said compositions. Moreover, the composition formed by pre-cured biopolymer 5 presents a decrease in viscosity when shear stress is increased, starting from viscosities of 10 Pa.Math.s and stabilizing at viscosities of 1 Pa.Math.s when the bio-ink is subjected to high shear velocities. This behavior of the bio-ink is similar to that described by a pseudoplastic fluid.

[0214] Furthermore, the composition comprising biopolymer 6 has a viscoplastic (or Bingham plastic) behavior, showing a viscosity of 2.35 Pa.Math.s until reaching a critical deformation stress corresponding to 248.6 1/s, after which viscosity starts to slightly decrease until reaching 1.41 Pa.Math.s.

[0215] It can be concluded that the compositions comprising monomer Y show a pseudoplastic behavior in the bio-inks. In the case of biopolymer 6, This behavior experiences a delay, which can start to be observed once a critical deformation stress has been exceeded. Said behavior corresponds to a plastic showing a viscoplastic or Bingham plastic behavior.

[0216] To simulate the injection process to which the compositions of the invention are subjected when they are used as bio-inks in a printer, a thixotropic analysis thereof was carried out. This analysis consists of subjecting the biopolymers to a high shear velocity for a short period of time, trying to mirror the forces to which the bio-inks are subjected when going through a needle having a very small diameter in the printing process.

[0217] The results obtained in the thixotropic analysis of the compositions described above clearly showed that there is no variation in their viscosity when the injection process is simulated, in other words, none of the analyzed compositions varied in viscosity when subjected to high shear stress for a short period of time (FIG. 28).

[0218] The variation in viscosity when an increase in temperature occurs was then analyzed. The analysis determines the ideal temperature at which the print bed should be pre-heated to achieve a higher viscosity in the bio-ink, and, therefore, a higher printing resolution.

[0219] The results demonstrate that the polymer viscosity of the analyzed compositions increases as the temperature increases until reaching a maximum value that depends on each composition, and it ranges between 18-22° C., and subsequently said viscosity gradually decreases as the temperature continues to increase (FIG. 29). This maximum viscosity reached is different for each bio-ink composition and said maximum is reached at a different temperature. Thus, as observed in FIG. 29, the composition comprising biopolymer 4 has a viscosity of 212.5 Pa.Math.s at a temperature of 15.0° C.; the composition comprising pre-cured biopolymer 5 has a viscosity of 90.9 Pa.Math.s at a temperature of 18.4° C.; the composition comprising biopolymer 6 has a viscosity of 371 Pa.Math.s at a temperature of 21.7° C.; and the composition comprising the mixture of biopolymer 4 (60% by weight)+pre-cured biopolymer 5 (40% by weight) has a viscosity of 242.6 Pa.Math.s at a temperature of 19.5° C.

[0220] Taking said results into account, there is a correlation between the maximum viscosities achieved by the studied bio-inks and their printability. Thus, bio-inks which show lower printability (bio-inks comprising biopolymers 4 and pre-cured biopolymer 5, respectively) reach a lower maximum viscosity than the biopolymers having a higher printability (bio-inks comprising the mixture of biopolymer 4 (60% by weight)+pre-cured biopolymer 5 (40% by weight) and the bio-ink comprising biopolymer 6). This is explained taking into account that the higher the viscosity a bio-ink demonstrates to present, the greater its printing fidelity will be.

[0221] Likewise, it has been observed that in all the bio-inks there is a critical temperature point after which viscosity starts to gradually increase, and said critical temperature point corresponds to about 8.5° C. for bio-inks comprising biopolymer 4 and the mixture of biopolymer 4 (60% by weight)+pre-cured biopolymer 5 (40% by weight), and 11° C. for bio-inks comprising pre-cured biopolymer 5 or biopolymer 6. This critical point indicates that in order to print with low viscosities, the bio-inks must be kept below this temperature in the reservoir.

Example 4. Fluid Stability of Bio-Inks

[0222] Next the stability of the compositions described in the present invention in a selected fluid was analyzed, said fluid being 1×PBS for the case of the present example. Given that the structures printed with the compositions described in the present invention will be used for in vitro cultures, or for tissue models, the structures must be able to remain stable in aqueous media for prolonged periods of time. For this example, cylinders are designed measuring 6 mm in diameter and 1.5 cm in height, and printing operations were carried out through a nozzle measuring 0.25 mm in diameter. The assayed compositions were the compositions comprising biopolymer 4, pre-cured biopolymer 5, biopolymer 6 or the mixture of biopolymer 4 (60% by weight)+pre-cured biopolymer 5 (40% by weight).

[0223] Printing with the composition comprising biopolymer 4 at a concentration of 250 mg/ml in 1×PBS shows good structural maintenance, but it is not capable of being maintained over time (FIG. 30). On the contrary, printing operations with the compositions comprising pre-cured biopolymer 5, under the same conditions as those mentioned above, show lower shape fidelity and collapsing thereof in a short time interval, which does not allow structures to be printed which suitably maintain their shape in height, despite showing stability over time (FIG. 31).

[0224] On the contrary, printing with the composition comprising the mixture of biopolymer 4 (60% by weight)+pre-cured biopolymer 5 (40% by weight) solves the drawbacks that said compositions present separately when used as bio-inks. This mixture allows printing showing good shape fidelity and structural maintenance over time to be performed, which allows complex structures to be made (FIG. 32). Likewise, the bio-ink comprising the composition with biopolymer 6 also shows structural maintenance over time, since it comprises monomers X and Y in its sequence (FIG. 33).

Example 5. Evaluation of Cell Viability and Cytotoxicity of the Compositions

[0225] Once the printability of the compositions of the invention as bio-inks and their consistency over time were evaluated, their cytotoxicity and cell viability were evaluated using human fibroblast cell line HFF-1. To this end, porous circular gratings are printed measuring 5 mm in diameter and 1 mm in height, with a square porosity of 1 mm sidewise.

[0226] The composition selected for performing the assays was the composition comprising the mixture of biopolymer 4 (60% by weight)+pre-cured biopolymer 5 (40% by weight), since, as demonstrated in the previous examples, these are the compositions which have demonstrated the best printability and stability over time, since they comprise monomers X and Y. To determine the bioactivity that said composition presents when it comprises in the sequence of biopolymers comprising monomer D, specifically monomer D comprising the RGD sequence, more specifically monomer D comprising the sequence SEQ ID NO: 6, a composition comprising the mixture of biopolymer 3 (60% by weight)+biopolymer 2 (40% by weight) is used as a negative control, wherein said composition comprises the same structure as the other assayed composition but lacks monomer D, which allows the functionalization and therefore the bioactivity of the compositions.

[0227] On each surface printed with each of the aforementioned compositions, 10,000 cells will be seeded. An Alamar blue assay is performed to study early cell viability and proliferation. Alamar Blue is a reagent containing a fluorescent indicator which is reduced by changing color as a result of cellular metabolic activity, allowing the quantitative determination of cell viability and cytotoxicity. Through the Alamar blue assay, early cell viability is observed at 30 minutes, 2 hours and 4 hours after seeding with each of the assayed compositions. The number of cells adhered on the surfaces is calculated by means of a calibration line, and it is clearly shown that the number of cells which adhere is significantly higher in those gratings which have been printed with the composition comprising the mixture of biopolymer 4 (60% by weight)+pre-cured biopolymer 5 (40% by weight) comprising monomer D, with respect to the composition comprising the mixture of biopolymer 3 (60% by weight)+biopolymer 2 (40% by weight), which does not comprise monomer D (FIG. 34). Therefore, it can be deduced that the presence of the RGD integrin adhesion sequence in bio-inks allows for and improves early cell adhesion.

[0228] Furthermore, fibroblast cell proliferation on the gratings printed with the aforementioned compositions for long periods of time, that is, 21 days, is also analyzed. The results show a gradual increase in the percentage of reduction of AlamarBlue in both compositions over time, starting from a percentage of 4.4% in the beginning up to 64.1% in the case of the composition comprising the mixture of biopolymers 4 (60% by weight)+pre-cured biopolymers 5 (40% by weight), and from 2.2% in the beginning up to 53.2% in the case of the composition comprising the mixture of biopolymers 3+(60% by weight)+biopolymer 2 (40% by weight) (FIG. 35). Said percentage is significantly higher in the case of gratings which have been printed with the composition comprising the mixture with monomer D in its sequence.

[0229] To demonstrate that the cells have adhered to the gratings printed with the aforementioned compositions, DAPI/Phalloidin staining was carried out. DAPI staining is used to stain the adenine-thymine bonds of the DNA present in the cell nucleus, while Phalloidin is used for staining actin filaments, allowing the observation of the rest of the cytoplasm. The combination of both staining techniques allows cell morphology to be observed.

[0230] DAPI/Phalloidin staining is performed 14 days after of culturing fibroblasts with gratings printed with the composition comprising biopolymer 4 (60% by weight)+pre-cured biopolymer 5 (40% by weight). In FIG. 36, it can be observed how the cells have adhered to the printed gratings, preferably being situated longitudinally, forming a three-dimensional matrix. The cells are also observed arranged at different heights corresponding to the deposition of the different fibers (FIG. 37), demonstrating that the morphology or structure of the grating conditions cell arrangement and growth.

Example 6. Evaluation of Cell Viability and Cytotoxicity of Cells Embedded in the Bio-Ink (Biopolymer 6) Prior to Bioprinting

[0231] Next, the cell viability and morphology shown by human fibroblasts HFF-1 when printed together with biopolymer 6 are evaluated.

[0232] To this end, HFF-1 cells (6×10.sup.6 cells/ml) are mixed together with the biopolymer 6 dissolved in DMEM and printed in porous circular gratings measuring 5 mm in diameter and 1 mm in height, with a square porosity of 1 mm sidewise. Once the surfaces have been printed under sterility, they are immersed in the cellular medium and incubated for 21 days.

[0233] First, a LIVE/DEAD™ staining is performed on the printed surfaces. Staining of this type serves to determine cell viability by means of staining live and dead cells. By obtaining photographs of different fields of the printed grating, cells can be counted and a percentage of viability (percentage of live cells) established. In order to know if cell viability is modified due to the printing process, control is performed, said control being a deposition of the same biopolymer and the same cellular concentration mixed and deposited on a grating without having been subjected to the 3D bioprinting process.

[0234] The analysis of the presence of live and dead cells is performed 4 hours after printing, and on different days: day 1, day 3, day 7, day 14 and day 21. In FIG. 38, a viability of 76% is observed in cells printed together with biopolymer 6 4 hours after being printed. However, viability values increased significantly after 7 days of culture, reaching 90% cell viability. During the first few days of cell culture, a noticeable difference is observed between the cell viability obtained in the printed gratings and their corresponding control. These results suggest that during the first few days of culture, the extrusion of biopolymer 6 negatively affects cell viability, although said long-term cell viability is not affected.

[0235] Cell morphology, reorganization and proliferation of HFF-1 were studied by means of light microscopy through DAPI/Phalloidin staining, explained above. This staining was performed after printing biopolymer 6 mixed together with cells (6×10.sup.6 cells/ml) after 1, 3, 7, 14 and 21 days. As observed in FIG. 39, from the first day, cells are homogeneously distributed on the printed surfaces, which denotes a good distribution of nutrients through the gratings which keeps the cells located on both the inner and outer parts thereof. In addition, during the first steps/days of culture, the cells remain round, but after the three first days of incubation, they start to develop an elongated and fibrous shape, which is characteristic of this cell type (fibroblasts), being completely spread out 7 days after culture. In this step, the cells started to form aggregates along the structures and maintained growth and proliferation for the remaining days of culture, up to 21 days, when the experiment ended.

[0236] Therefore, as shown in the examples included herein, the compositions comprising the mixture of biopolymer 4+pre-cured biopolymer 5, as well as the compositions comprising biopolymer 6, can be used as bio-inks. Said compositions present good printability, allowing structures that are resolute in height and stable over time to be printed (Table 9, FIGS. 30 and 31), specifically as a result of the presence in the sequences thereof of monomers X and/or Y. In addition, they present low viscosities which facilitate printing at low temperatures (FIG. 27), and a rapid increase in viscosity when temperature increases (FIG. 29), which facilitates the printed structure remaining stable in the printing process. Additionally, they allow cell adhesion and proliferation as a result of the presence of monomer D.