Collagen ink for 3d printing

Abstract

The present invention relates to a collagen ink for 3D printing. The present invention also relates to a process for obtaining said ink and uses conferred thereto.

Description

[0001] This invention relates to collagen ink for 3D printing. The invention also relates to methods for obtaining said ink and its intended uses. Background Technology

[0002] 3D printing is a widely used technology in the fields of biology and medicine, in which biological constructs are obtained to serve regenerative medicine and biological research.

[0003] Ink used for 3D printing must possess sufficient physical properties, such as viscosity, elasticity, porosity, and stability, but it must also be compatible with various applications, such as tissue development, and therefore must possess sufficient biological properties. The ink must have sufficient flowability to be extruded through the printer nozzle, and the printed construct must remain intact over time at physiological pH levels.

[0004] Examples of materials used in inks for biological applications include alginate, fibrous protein, and collagen.

[0005] Collagen is one of the most abundant proteins in nature and is responsible for maintaining the structural integrity of tissues. Collagen is the most widely used material in cellular applications because it is the most abundant component of the extracellular matrix; this material is used, for example, as a carrier matrix to aid cell growth. Collagen fibers possess certain amino acid sequences that allow cell adhesion and proliferation. Collagen matrices are extremely useful and have led to several important biological advances; however, an optimal 100% collagen matrix has not yet been developed. Components composed solely of collagen are made from soluble collagen and generally do not possess the consistency and stability sufficient to maintain the integrity of printed constructs without the aid of cross-linking agents, or they lack a natural conformation.

[0006] In the case of collagen, to give it sufficient viscosity and stability for use as ink, it can be presented in the form of a hydrogel. An example of this can be found in patent publication number CN106581753, which describes a bio-hydrogel for 3D printing of a matrix for skin tissue growth. The hydrogel contains 1% to 15% nanocrystalline cellulose, 65% to 98% collagen, and 0.01% to 20% crosslinking agent. This type of hydrogel has a viscosity suitable for use as ink, but if a crosslinking agent is not added to the collagen to enable it to function, the composition obtained from the hydrogel will lose its integrity.

[0007] To endow collagen with the necessary properties for use as a 3D printing ink in biological applications, a strategy of functionalizing collagen is employed. Patent publication CN106237383 describes an example of functionalized collagen microfilaments containing growth factors that promote cell growth and differentiation. In this case, the fibers are also sorted and pre-selected to obtain very small-sized fibers, the size of which is adjusted to be smaller than the diameter of the extruder needle (e.g., the inner diameter of a 27G needle is 210 μm), based on soluble collagen extracted with surfactants such as SDS and triton, and further molecularly modified to obtain optimal results. Furthermore, rheological data or stability of the obtained ink, its printability, or the integrity of the obtained composition are not shown. In the patent, fibers of 100 μm, 75 μm, 38 μm, and up to 25 μm are selected.

[0008] Another strategy already used to produce collagen matrices for 3D printing is cryogenic printing, which allows for the deposition of low-viscosity collagen solutions at sub-zero temperatures and in-situ cross-linking.

[0009] Current extraction and purification methods for collagen significantly reduce its cross-linking density, thereby affecting related properties and biological responses.

[0010] Therefore, based on existing technology, there is a need to develop inks based on fibrous collagen, which are not denatured and have sufficient consistency and stability to be used as inks in 3D printing without the aid of crosslinking agents. Summary of the Invention

[0011] Collagen is the most abundant protein in the extracellular matrix of connective tissues such as the skin, bones, cartilage and tendons of mammals, and accounts for more than 90% of their dry weight.

[0012] The basic unit of collagen consists of three polypeptide chains that appear to be entangled together, forming a triple helix, thus constituting a large molecular unit called procollagen. Procollagen molecules aggregate to form collagen fibers.

[0013] In this invention, a collagen ink for 3D printing was developed, which has a viscosity suitable for 3D printers and maintains collagen in its natural state. The ink of this invention is characterized as a dispersion of natural collagen fibers in an aqueous acidic medium.

[0014] An acidic medium allows collagen to absorb water and produce a squeezeable fluid.

[0015] Therefore, a first aspect of the present invention relates to a collagen ink for 3D printing, comprising a dispersion of natural collagen fibers in an acidic medium at a weight concentration of 0.1% to 10% and a pH of 0.5 to 5, the collagen ink for 3D printing having a viscosity of 10 centipoise (cP) to 50 million centipoise (McP) (Brookfield method, at a temperature of 18°C ​​to 22°C), and wherein the fiber dispersion comprises fibers having a length in the range of 1 μm to 2500 μm as measured at a pH of 1 to 2, and a diameter in the range of 0.1 μm to 180 μm.

[0016] The fibers in the ink of this invention have a wide range of lengths and diameters, and there is no need for sorting to obtain and select small-sized ink fibers. The ink is printable even if it may have a high percentage of large fibers in its mass (where the fiber length is greater than 1 mm and the diameter can be greater than the inner diameter of the printer extruder head, for example, 22G = 152 μm).

[0017] The collagen fibers of this invention retain their triple helix molecular structure without denaturation. The fibers can absorb water and swell, with a diameter primarily between 5 μm and 35 μm, while they will not exist under denaturation and hydrolysis.

[0018] As already mentioned, collagen molecules are elongated molecules composed of three polypeptide chains entangled together, forming a triple helix stable by hydrogen bridging or interaction—the structure of natural collagen. When collagen denatures, for example by heating it in the presence of water or heating it to extreme pH values, these chains separate and dissolve, thereby forming gelatin. Gelatin formation is due to the breaking of the hydrogen bonds that stabilize the collagen triple helix; the process of collagen transforming into gelatin is considered a typical denaturation process. In the dispersion of collagen fibers of the present invention, there is no gelatin or the presence of gelatin is negligible.

[0019] In this specification, "natural collagen" should be understood as collagen that has not been completely or significantly denatured, hydrolyzed, or gelled.

[0020] The ink of the present invention is presented as a dispersion of optimized-size natural fibrous collagen in an acidic medium. The natural fibrous collagen is obtained from a biological source of collagen and typically consists of treated large bundles and / or fibrous aggregates. The treatment preserves the natural structure and induces the breakdown into fibers of smaller diameter and length, thereby allowing the formulation of fluids with rheological properties suitable for proper 3D printing.

[0021] The structures obtained by 3D printing using the ink of this invention are stable and resistant structures of natural, insoluble fibrous collagen without the need for cross-linking agents.

[0022] Importantly, the ink contains natural collagen, because in nature, collagen is not only a structural and carrier protein, but it also interacts specifically with other biomolecules to mediate cell adhesion and guide cell function. As long as the ink of the present invention retains its natural structure, it will be able to maintain its properties related to its physiological function when used in printing for biomedical applications.

[0023] Similarly, the present invention relates to any hydrogel that incorporates the ink described in the first aspect of the invention and the embodiments described below.

[0024] A second aspect of the present invention is a method for obtaining collagen ink, the method comprising the following steps:

[0025] a) Wash and cut the collagen-containing tissue;

[0026] b) Chemical impregnation of the severed tissue;

[0027] c) Wash the product obtained in step b) with water;

[0028] d) Adjust the pH of the product obtained in c) to a value between 0.5 and 5 to cause the product from step c) to swell:

[0029] e) Mechanically grind the product from step d);

[0030] f) Disperse in water to a concentration preferably 0.1% to 10% by weight.

[0031] The collagen matrix exhibits low immunogenicity, good biocompatibility, and biodegradability, interacts specifically with other biomolecules, and contains specific sequences that mediate the regulation of cell morphology, adhesion, migration, and differentiation. The ink of this invention has a viscosity suitable for passing through a 3D printing nozzle.

[0032] Therefore, a third aspect of the present invention relates to the use of the ink of the present invention in 3D printing. A method for printing using the ink described above and in any of its embodiments is also described in the present invention.

[0033] A fourth aspect of the invention relates to a structure comprising the ink of the invention. One advantage of this structure is its high biocompatibility and absorbability. Attached Figure Description

[0034] Figure 1 A mesh printed with the ink of the present invention is shown as a biodegradable scaffold for cell culture.

[0035] Figure 2 The image shows the result obtained on day 2 after two days of cultivation. Figure 1 A culture (10×) containing a mesh of mouse embryonic fibroblasts within a reticulated structure.

[0036] Figure 3 The culture on day 5 is shown.

[0037] Figure 4 The culture is shown on day six of development.

[0038] Figure 5 Stress scan diagrams of samples 531-010 and 531-020 are shown.

[0039] Figure 6 Stress scan diagrams of samples 431-010 and 431-020 are shown.

[0040] Figure 7 The composition printed using the ink of the present invention is shown.

[0041] Figures 8A to 8F Different compositions made with the ink are shown. Figure 8B 431-010 and 8F correspond to the constituents A, C and E after neutralization and adjustment to physiological pH (pH 7.4).

[0042] Figures 9 and 9B show optical microscopic images of individual particles of the ink 531-010 of the present invention diluted to 0.1% and stained with Sirius red.

[0043] Figure 10 An element printed using the ink of Example 8 is shown. Detailed Implementation

[0044] As mentioned above, a first aspect of the present invention relates to a collagen ink for 3D printing, comprising a dispersion of natural collagen fibers in an acidic medium at a concentration of 0.1% to 10% and a pH of 0.5 to 5, wherein the viscosity of the collagen ink for 3D printing is 10 cP to 50 McP (according to the Brookfield method described in Example 3 and at a temperature of 18°C ​​to 22°C), and wherein the fiber dispersion comprises fibers having a length in the range of 1 μm to 2500 μm measured at a pH of 1 to 2, and a diameter in the range of 0.1 μm to 180 μm.

[0045] Preferably, 90% by mass of the collagen fibers in the dispersion comprises fibers with a length ranging from 50 μm to 2500 μm.

[0046] Preferably, 75% by mass of the collagen fibers in the dispersion comprises fibers with a length ranging from 50 μm to 1000 μm, and 50% by mass of the collagen fibers comprises fibers with a length ranging from 100 μm to 500 μm.

[0047] Preferably, 80% of the collagen fiber mass comprises fibers with a diameter of 5 μm to 35 μm.

[0048] Preferably, the concentration is 1% to 5%.

[0049] Preferably, the pH is between pH 1 and 3.

[0050] Preferably, the viscosity is 2 McP to 15 McP.

[0051] The collagen matrix of the present invention is characterized by its integrity after being neutralized to physiological pH.

[0052] The characteristic of the collagen matrix of the present invention is its integrity after neutralization to physiological pH. One possible application of the ink of the present invention is printing matrices for cell and tissue culture. In this case, it is highly advantageous that the collagen is natural, as it more closely resembles the original structure of collagen in this way, thereby replicating the original environment in which cell proliferation occurs in vivo with greater similarity. Surprisingly, the resulting composition or matrix is ​​rigid enough to remain intact and stable over time at physiological pH. Preferably, the ink used in these applications comprises a dispersion of natural collagen fibers at a concentration of 2% to 5% in an acidic medium and a pH of 1 to 3.

[0053] The preferred use of ink, as described in the previous paragraph, is in constructing substrates for cell and tissue culture.

[0054] As described above, a second aspect of the present invention relates to a method for obtaining the ink of the present invention from collagen-containing tissue, the method comprising the following steps:

[0055] a) Wash and cut the collagen-containing tissue;

[0056] b) Chemical impregnation of the severed tissue;

[0057] c) Wash the product obtained in step b) with water;

[0058] d) Adjust the pH of the product obtained in c) to a value of 0.5 to 5 to cause the product in step c) to swell;

[0059] e) Mechanically grind the product from step d);

[0060] f) Disperse in water to a concentration preferably from 0.1% to 10%.

[0061] Preferably, the collagen-containing tissue is connective tissue. Connective tissue is preferably selected from: dermis, bone, tendon, cartilage, and intestine. More preferably, the connective tissue is dermal tissue, and even more preferably, the collagen is extracted from a layer called the dermis.

[0062] Regarding the source of the connective tissue, it can be from any animal source. In one specific embodiment, the connective tissue is from cattle, sheep, pigs, birds, fish, or a mixture thereof. Preferably, the connective tissue is from cattle and from animals older than one year old. Preferably, it is from one to three years old.

[0063] More preferably, when the connective tissue is dermis, it is depilated and bleached before step a). In addition to washing step a), a bleaching step using a weak oxidizing agent is also preferred. An example of a weak oxidizing agent is dilute hydrogen peroxide.

[0064] Step b) is preferably carried out in the presence of an alkaline reagent, such as calcium hydroxide or sodium hydroxide, with or without an enzyme. In particular, step b) is carried out using a sulfur salt and an alkali, such as Na₂S and Ca(OH)₂.

[0065] Preferably, the acid used for acidification in step d) is selected from hydrochloric acid, lactic acid, acetic acid, and citric acid. Preferably, the acidification is carried out to a pH of 1 to 3.

[0066] Preferably, step f) for preparing the aqueous dispersion of fibrous collagen is carried out at a concentration of 1% to 5%. More preferably, 2% to 5%.

[0067] Specifically, it is preferable to subject the dispersion obtained in step f) to a mechanical homogenization process, which includes subjecting the dispersion to shear forces by passing it through a slit under high pressure, preferably at a minimum of 50 atmospheres. Preferably, the pressure is between 50 and 100 atmospheres.

[0068] In Example 3 and Figure 5 and Figure 6 The diagram illustrates the rheological changes in the ink of the present invention caused by the homogenization process. The homogenized material can withstand greater forces without losing its structure. When a greater deformation force is applied than that in the unhomogenized material, the elastic modulus (G') of the collagen material remains constant.

[0069] The present invention also relates to products obtained by the method of the present invention and by all embodiments thereof described in the preceding paragraphs.

[0070] The method of printing with the ink of the present invention has different implementations.

[0071] In one embodiment of the printing method, the ink is mixed with cells, specifically selected from: astrocytes, embryonic cardiomyocytes, fetal cardiomyocytes, neonatal cardiomyocytes, cardiomyocytes, embryonic ventricular myocytes, corneal endothelial cells, corneal epithelial cells, iris pigment epithelial cells, retinal pigment epithelial cells, fetal dopamine neurons, fetal neocortical neurons, enteric neurons, hepatocytes, adipose tissue mesenchymal stem cells, bone marrow mesenchymal stem cells, osteoblasts, chondrocytes, pancreatic cells, and urothelial cells.

[0072] In another embodiment of the printing method, prior to printing, the ink is neutralized to a pH preferably 7 to 8, specifically by neutralizing it with a salt or buffer selected from: phosphate-buffered saline (1× and 10× PBS), Tricine, MOPS (3-(n-morpholino)propanesulfonic acid), HEPES (4-2-(hydroxyethyl)piperazine-1-ylethanesulfonic acid), tris, and sodium carbonate.

[0073] Example

[0074] The following embodiments are merely examples of the present invention and should not be construed as limiting the present invention.

[0075] Example 1

[0076] To prepare the ink of the present invention, 18- to 26-month-old cowhides are used and subjected to a standard hair removal process (e.g., with sulfur salts such as Na2S).

[0077] Next, the skin is divided and the dermis is extracted to obtain collagen.

[0078] After washing several times with water and bleaching with a mild oxidizing agent (e.g., dilute hydrogen peroxide), the material is trimmed with calcium hydroxide under the required time and temperature conditions for full impregnation. The reagent residue is then washed again with water and acidified with concentrated HCl (33%). The mixture is then ground using a grinder and diluted with water until a dispersion of 2.9% collagen in water is obtained. The resulting pH is 3.1.

[0079] Example 2

[0080] The dispersion of Example 1 was printed using a 3D printer with a 24G nozzle needle extruder at a speed of 3 mm / s. A mesh matrix (5 cm × 2.5 cm with 1.2 mm pore size) was printed in a 10 cm culture dish as a scaffold for use as a substrate for cell culture.

[0081] After printing the mesh, it is immersed in 50 mM NaOH to cover the mesh, thereby raising the pH and causing the collagen to coagulate. After coagulation, it is washed with a sufficient amount of phosphate-buffered saline (PBS) at pH 7.4 to cover the mesh, and then immersed in an antibiotic-rich culture medium. It should be noted that this dispersion of the present invention is printed and coagulated at room temperature without the addition of a crosslinking agent.

[0082] Mouse embryonic fibroblasts were grown on a mesh. A total of 300,000 cells were resuspended.

[0083] Figure 2 The culture containing a mesh of fibroblasts is shown after 2 days. Figure 3 The image shows the culture on day five. The culture plate is at 100% confluence, and cell tactisms and directed growth are beginning to be observed. Figure 4 The culture is shown on day six of development. Some cells are growing along the direction of collagen fibers in the network.

[0084] Example 3: Rheology of Ink

[0085] For this purpose, a controlled stress rheometer, the "Modulate Advanced Rheometer System" MARS 40 (Thermo Haake), equipped with a 20 mm plate-to-plate rotor, was used. Maintaining a 0.8 mm gap between the plates, the stress was measured at a fixed frequency of 1 Hz at a measurement temperature of 15.0 °C within a stress range of 1 Pa to 15000 Pa. An oscillatory stress scan test was performed inside. Before the test, after reaching a gap of 0.8 mm, the sample was tempered between the plates for 10 minutes to ensure the measurement temperature.

[0086] Stress scanning at a fixed frequency allows for the identification of linear viscoelastic regions.

[0087] Oscillation measurements in the linear viscoelastic region are very important because the material’s response will depend only on its structure and not on rheological parameters such as stress or deformation.

[0088] During testing, the values ​​of G' and G (vertical axis) are expressed as functions of stress (horizontal axis). The cross point, LVR, and flow limit value correspond to stress, i.e., the horizontal axis.

[0089] The elastic modulus or storage modulus (G') is related to the energy stored in the material, while the viscous modulus or loss modulus (G”) is related to the energy consumed by the material.

[0090] The intersection point is where G' and G” are equal. It has the same oscillatory shear stress value as the intersection point of the G' and G” curves, and therefore it is the stress value at which the material stops behaving as an elastic material and begins to behave as a viscous fluid.

[0091] The cross point value of homogenized material is about 40% higher than that of diluted material, which means that the material maintains its elastic behavior under higher stress and is therefore able to withstand greater stress without losing its structure.

[0092] Stress scanning allows for the determination of a material's yield point. When a material is not flowing (solid behavior), the elastic modulus remains constant, but it decreases as the material begins to flow. The value of the elastic modulus at which the material begins to flow is the flow limit, representing the point at which the material begins to undergo permanent deformation. Before this point, the deformation suffered by the material is recoverable, and therefore the values ​​of G' and G" are constant. However, from the flow limit onward, the material begins to lose its elastic components, and the viscous components increase until they become equal at the crossover point, at which point the material begins to behave like a viscous fluid. Figure 5 and Figure 6 Stress scan diagrams of samples 531-010, 531-020 and 431-010, 431-020 are shown.

[0093] Samples 531-010 and 431-010 are equivalent to Example 1, but with a collagen concentration of 4.8 ± 0.1%. In the case of samples H (431-020 and 531-020), they are the same dispersions, but after undergoing a high-pressure homogenization process in an industrial homogenizer, which essentially involves forcing the material through a slit at pressures ranging from 50 to 300 atmospheres using a piston.

[0094] The diluted and homogenized materials 531-020 and 431-020 have higher flow limits than the diluted and unhomogenized materials 531-010 and 431-010, and therefore can withstand greater stress, thus maintaining the integrity of their elastic components and ultimately preserving their structural properties.

[0095] Table 1. Rheological data of inks 531-010, 431-010, 531-020, and 431-020 of the present invention after stress scanning at a frequency of 1 Hz.

[0096]

[0097] * Corresponds to the values ​​of G' and G” during the first 100 seconds of the analysis, thus ensuring that it is in the linear phase of the curve.

[0098] A rapid method for quantifying the extent of degradation and hydrolysis of natural collagen is to determine the soluble fraction at 10% ammonium sulfate after centrifugation at 18,000 rpm. The collagen content in this fraction is then quantified using the biuret method, and the result is expressed as a percentage of the collagen percentage previously quantified using the biuret method. This fraction is particularly present in degraded collagen.

[0099] Subsequently, the collagen content in the fractions was quantified using the biuret method, and the results are expressed as a percentage (Table 2).

[0100] Table 2. % solids and soluble fractions.

[0101]

[0102] ND: Not detected

[0103] The viscosity of the dispersion of the present invention was measured.

[0104] The viscosity of the ink of this invention is determined using a Brookfield DV2T HBT viscometer (Brookfield Ametek). The viscosity is calculated from the torque measured based on the selected shaft and rotational speed.

[0105] The shaft used in this method is an inverted T-shaped shaft (Helipath with a T-shaped rod shaft). The following are the viscosity ranges measured using the various shafts for this viscometer model.

[0106] T-shaped shaft Axis number SMC viscosity (cP) TA 91 2016.000-16.000.000 TB 92 4032.000-32.000.000 TC 93 10080.000-80.000.000 TD 94 200160.000-160.000.000 TE 95 500400.000-400.000.000 TF 96 1000800.000-800.000.000

[0107] *SCM (Spindle Multiplier Constant) is a characteristic constant for each shaft used to convert torque measurements into viscosity.

[0108] Other axes are used for substances with lower viscosity ranges and collagen concentrations equal to or less than 2%.

[0109] axis Viscosity range (cP) HB-1 2.096-20.960 HB-2 8.880-88.800 HB-3 42.800-428.000 HB-4 434.400-4.344.000 HB-5 170.400-1.704.000

[0110] The device is configured in such a way that it requires the specification of the shaft and rotational speed used, and it performs the conversion to viscosity in cP.

[0111] For this purpose, the substance was placed in a 250 ml beaker. The temperature of the substance was maintained between 18°C ​​and 22°C. The appropriate shaft was used according to the expected viscosity of the substance. Therefore, 94 was used for the homogenized substance (531-020), while 95 was used for the unhomogenized substance (531-010). The rotation speed was 1.0 RPM, and 250 measurements were taken over a period of 4 minutes.

[0112] The viscosity measurements after homogenization were significantly reduced, resulting in 23.3 McP for 531-010 and 6.5 McP for 531-020 (Table 1). This reduction in viscosity greatly improves the printability of the ink and reduces the pressure required to print the ink using a printer.

[0113] Example 4. Effect of pH on ink

[0114] In this context, the behavior of the ink of the present invention and the neutralized ink of the present invention were compared. It is important to maintain the structure and stability of the material after neutralization to physiological pH, ensuring that the structure and shape of the 3D construct to be printed will remain sufficiently stable and stable under the physiological conditions required for use in biomedical applications, where they will be used as carriers for cell growth. As can be seen from the table below (Table 3), the G' values ​​of the neutralized materials (neutral 531-010 and neutral 531-020) remain high despite a decrease in elastic modulus. Therefore, after neutralization to physiological pH with different bases and placement in media similar to cell culture media such as PBS, calcium-rich Hank solution (HBSS-Ca), or equivalents, the materials remain sufficiently stable over time.

[0115] Rheological analysis was performed by frequency scanning, which included oscillation testing under a fixed stress of 100 Pa, with frequency scanning ranging from 0.01 Hz to 100 Hz, a plate gap of 0.8 mm, and a measurement temperature of 15 °C. The values ​​obtained for a frequency of 0.1 Hz are shown in the table below, where the stability of the material of the present invention in Example 3 with a collagen concentration of 4.8% can be observed; after neutralization to physiological pH with NaOH (0.05 M) and equilibration in PBS, it maintains a very high elastic modulus (G').

[0116] Table 3. Rheological parameters of the ink of the present invention before and after neutralization to neutral pH. Values ​​at a frequency of 0.1 Hz.

[0117]

[0118] In the same manner, the stability of the neutralized material over time was determined. For this purpose, another batch (batch 6) of the gel of the present invention prepared as Example 3 was neutralized with NH4OH (0.25%) or NaOH (0.05M) and adjusted to physiological pH with HBSS-Ca. Approximately 0.3 g of the sample was taken and a neutralizing agent was added to cover it.

[0119] As shown in the table below, after neutralization with NaOH and washing and equilibration with PBS at physiological pH, the material maintained a high and stable elastic modulus over time, despite an initial decrease in elastic modulus. This confirms that its structure was maintained under physiological conditions (37°C and pH 7.4) comparable to those used for cell culture. Similar observations were obtained with other bases, such as ammonium hydroxide, and other equilibrated media, such as HBSS-Ca, confirming the maintenance of a stable constituent shape in common media.

[0120] Table 4 shows the evolution of the storage modulus or elastic modulus (G') of the neutralized sample 231-020 over time. Value at 0.1Hz.

[0121] G'(Pa) Days alkali 4675 0 NaOH 3242 7 NaOH 3038 14 NaOH

[0122] Example 5. Printing using the ink from Example 3

[0123] The pressure of the equipment was adjusted accordingly (550 kPa) and printing was performed using a 22G nozzle needle extruder at an extrusion speed of 2 mm / s. Although the ink contains insoluble collagen fibers, the continuity of the composition was identified, such as... Figure 7 As seen in the text.

[0124] Example 6. Printability of the ink of the present invention.

[0125] Figures 8A to 8F An example of a structure obtained by adjusting the pressure of the 3D printer accordingly to an extrusion speed of 5 mm / s using the ink of Example 3 is shown. The continuity of the structure is identified.

[0126] Figures 8E to 8F Corresponding to multi-layered 3D structures.

[0127] Figure 8B , 8D 8F corresponds to the same construct on the left, which was neutralized with 50 mM NaOH and adjusted to pH 7.4 (physiological pH, the pH suitable for optimal cell proliferation) with PBS. In all cases, the integrity of the construct was maintained, and its use was permitted for applications such as those previously shown.

[0128] When the ink of the present invention is prepared at a concentration of less than 1%, a significant decrease in the stability of the composition is observed, with a viscosity value of 6,000 cP for an ink containing 1% collagen. Therefore, these conditions are not optimal when the composition or matrix needs to be neutralized, but the ink can be useful when the printed construct does not require neutralization.

[0129] This can happen in a similar way if the ink's pH drops excessively. For example, when the ink's pH is 0.6, the 531-020 ink experiences a decrease in elasticity due to a drop in G' value to 3900 Pa and a decrease in viscosity to 3.14 McP. After printing, the composition loses its stability during the neutralization process.

[0130] During the acidification process in step d) of the production process, hydrochloric acid, lactic acid, or acetic acid is used to produce fully printable ink that maintains the integrity of the composition after neutralization at the pH and concentration values ​​mentioned above.

[0131] Example 7. Morphological Analysis

[0132] Morphological analysis was performed using dynamic image analysis of inks 531-010 and 531-020 from Example 3. For this purpose, the inks were diluted to approximately 1% in deionized water and the pH was adjusted to approximately 1.5 with 1M HCl. The diluted samples were vortexed at 2500 rpm for 10 minutes and then diluted to 1 / 20 with deionized water.

[0133] Measurements were performed on a Sympatec QICPIC / Lixell particle analyzer with a continuous flow tray (0.5 mm wide) and a particle size range of 4 μm to 2888 μm. Due to the highly heterogeneous particle distribution typical of collagen suspensions, at least three separate measurements were performed on each of the two subsamples. A total of 1 million to 3 million particles were detected in each sample.

[0134] Evaluation was conducted based on fiber length and diameter distribution. The proportion of fibers of a certain length or width in the total mass of collagen fibers in the sample was calculated. Tables 5 and 6 show the length and diameter (in μm) of collagen fibers with a cumulative distribution percentage of 10%, 16%, 50%, 84%, and 90% of the total mass, calculated from the average value.

[0135] Table 5. Fiber length (μm) corresponding to 10%, 16%, 50%, 84%, and 90% of the total collagen fiber mass in inks 531-010 and 531-020 of the present invention.

[0136] 10％ 16％ 50％ 84％ 90％ 531-010 244 336 834 1775 2291 531-020 99 135 383 989 1235

[0137] Table 6. Fiber diameter (μm) corresponding to 10%, 16%, 50%, 84% and 90% of the total mass of collagen fibers in inks 531-010 and 531-020 of the present invention.

[0138] 10％ 16％ 50％ 84％ 90％ 531-010 9.3 11.6 19.0 27.0 30.4 531-020 10.2 12.3 19.2 27.6 30.8

[0139] As seen in Table 5, sample 9D exhibits a wider fiber length distribution. The median is 838 μm. This means that 50% of the total collagen fiber mass in the sample is provided by fibers longer than 838 μm. 84% of the collagen protein mass is provided by fibers longer than 336 μm. However, in 531-020, the proportion of smaller fibers is much higher. The median of 383 μm is significantly lower than that of sample 531-010. This means that compared to 838 μm in sample 531-010, half of the collagen protein mass in sample 531-020 is composed of fibers shorter than 383 μm.

[0140] Therefore, the fiber size shortens with homogenization, which helps the ink pass through the extruder and ultimately contributes to its printability, thus reducing the pressure required to print it with a commercial printer.

[0141] The cumulative distribution of fiber diameters (Table 6) of samples 531-010 and 531-020 was almost identical, with a median of 19 μm and 80% of the fibers having diameters ranging from 10 μm to 31 μm.

[0142] Figure 9A and 9B An optical microscope image of a single particle of the ink 531-010 of the present invention, diluted to 0.1% and stained with Sirius red, is shown. The image allows for a visual image of the fibers present in the ink of the present invention, in which a broad distribution of fiber length and diameter is clearly observed.

[0143] Example 8

[0144] Prior to printing, the acidic ink from Example 1 was neutralized, but at a concentration of 5%, and mixed with 1M tris(hydroxymethyl)aminomethane (Tris) adjusted to pH 7.4 at a 3:2 (V:V) ratio to obtain a 3% concentration. This ratio can be varied to obtain a more diluted ink. Similarly, half of the buffer can be replaced with a culture medium such as DMEM (Dulbecco's Modification of Eagle's Medium). For mixing, one syringe was filled with 3 ml of collagen and the other with 2 ml of Tris. The two syringes were connected using a connector, and the contents were transferred from one to the other, repeating this final step 40 times to ensure uniform mixing. Subsequently, the pressure was adjusted to 70 kPa, and printing was performed using a 20G nozzle needle extruder at an extrusion speed of 5 mm / s. The resulting prints are shown below. Figure 10 middle.

[0145] Example 9. Cell encapsulation in neutral collagen ink

[0146] The cell density figures presented in this embodiment are based on studies of mouse fibroblasts. The method used to mix the different components in this embodiment is similar to the method used in Example 8 for neutralized ink.

[0147] As mentioned in Example 1, collagen material was obtained at neutral pH. In this case, the amount required for subsequent mixing with cells was calculated based on the amount of collagen material mixed with the corresponding buffer at acidic pH. For this purpose, three parts collagen, one part buffer, and one part DMEM (Dulbecco modified Eagle medium) medium were mixed with cells. In this case, the acidic collagen material was mixed with the corresponding buffer 40 times (as mentioned in the previous example of neutralized collagen), and then mixed with the cell-containing medium in the same manner, but this time for a total of 20 mixing times.

[0148] To obtain a culture medium containing cells, a normal cell harvesting protocol is used to obtain a suspension culture medium with a density of approximately 200,000 cells / ml.

[0149] After all components were mixed, the cells were bioprinted onto plates prepared for cell culture. Following bioprinting, enriched DMEM was added to cover the entire scaffold or construct. Cell viability testing (live / dead assay) was performed 5 days after cell bioprinting. To complement this study, the scaffold (carrier) was digested using a combination of trypsin-EDTA and collagenase to obtain cells (both cells on the surface and cells embedded in the ink) (first incubation with 0.05% trypsin-EDTA for 20 minutes; then a second incubation with 500 U / ml collagenase until the scaffold was completely dissolved). Results showed cell viability greater than 90%.

Claims

1. A collagen ink for 3D printing comprising a dispersion of native collagen fibers in an acidic medium at a concentration by weight comprised between 0.1% and 10% and at a pH comprised between 0.1 and 5, the viscosity of the collagen ink for 3D printing measured by the Brookfield method at a temperature comprised between 18°C and 22°C is comprised between 2 McP and 15 McP and wherein 75% of the mass of collagen fibers of the dispersion comprises fibers having a length measured at a pH comprised between 1 and 2 in the range 50 pm to 1000 pm and wherein 80% of the mass of collagen fibers comprises fibers having a diameter measured at a pH comprised between 1 and 2 comprised between 5 pm and 35 pm.

2. The collagen ink for 3D printing according to claim 1, characterized by 50% of the mass of collagen fibers of the dispersion comprises fibers having a length in the range 100 pm to 500 pm.

3. The collagen ink for 3D printing according to any one of claims 1 to 2, characterized in that... the concentration by weight is comprised between 1% and 5%.

4. The collagen ink for 3D printing according to any one of claims 1 to 2, characterized by the pH is comprised between 1 and 3.

5. A hydrogel comprising the collagen ink according to any one of claims 1 to 4.

6. A method for obtaining the collagen ink according to any one of claims 1 to 4 from a collagen-containing tissue comprising the following steps: a) washing and cutting the collagen-containing tissue; b) chemically impregnating the cut tissue in the presence of a calcium hydroxide, sodium hydroxide alkaline reagent treatment, or with a combination of Na2S and Ca(OH)2; c) washing the product obtained from step b) with water; d) adjusting the pH of the product obtained in c) to a value comprised between 0.5 and 5 to swell the product of step c): e) mechanically mincing the product of step d); f) dispersing in water to a concentration by weight comprised between 0.1% and 10%.

7. The method of claim 6, wherein the collagen-containing tissue is a connective tissue.

8. The method of claim 7, wherein the connective tissue is a dermal tissue of a layer called the dermis.

9. The method of any one of claims 7-8, wherein the connective tissue is derived from a bovine of an age comprised between 1 year and 3 years.

10. The method according to any one of claims 6 to 8, characterized in that in step d), the product of step c) is acidified to 1 to 3.

11. The method according to any one of claims 6 to 8, characterized in that the aqueous dispersion of fibrous collagen is carried out at a concentration by weight comprised between 1% and 5%.

12. Use of the collagen ink according to any one of claims 1 to 4 in printing a 3D construct.

13. A construct comprising the collagen ink according to any one of claims 1 to 4.

14. A method of printing using the collagen ink according to any one of claims 1 to 4, characterized by the method comprises the step of mixing the collagen ink according to any one of claims 1 to 4 with cells before printing.

15. The method of claim 14, wherein the cells are selected from the group consisting of: astrocytes, cardiomyocytes, corneal endothelial cells, corneal epithelial cells, iris pigment epithelial cells, retinal pigment epithelial cells, fetal dopaminergic neuronal cells, fetal neocortical neuronal cells, enteric neuronal cells, hepatocytes, adipose tissue mesenchymal stem cells, bone marrow mesenchymal stem cells, osteoblasts, chondrocytes, pancreatic cells, uroepithelial cells.

16. The method of claim 14, wherein the cells are selected from the group consisting of: embryonic cardiomyocytes, fetal cardiomyocytes, neonatal cardiomyocytes.

17. The method of claim 14, wherein the cells are selected from embryonic ventricular cardiomyocytes.

18. A method of printing the collagen ink according to any one of claims 1 to 4, characterized by the method comprises the following steps: neutralizing the collagen ink to a physiological pH comprised between 7 and 8; printing a construct.

19. The method of claim 18, wherein mixing the ink with cells.

20. The method of claim 18, wherein The neutralization is performed using a salt or buffer selected from the group consisting of phosphate buffered saline, Tricine, MOPS, HEPES, Tris, sodium carbonate. The neutralization is performed using a salt or buffer selected from the group consisting of phosphate buffered saline, Tricine, MOPS, HEPES, Tris, sodium carbonate.

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