Collagen ink for 3D printing

Abstract

The present invention relates to a collagen ink for 3D printing. The present invention also relates to a method for obtaining the ink and its use.

Description

Technical Field

[0001] The present invention relates to a collagen ink for 3D printing. The present invention also relates to a method for obtaining the ink and its use.

Background Art

[0002] 3D printing is a technology widely used in the fields of biology and medicine, providing biological structures for regenerative medicine and biological research.

[0003] Inks used for 3D printing need to have sufficient physical properties, for example, in terms of viscosity, elasticity, porosity, and hardness. They also need to be compatible with various applications, such as tissue development, and thus need to have sufficient biological characteristics for that purpose. The ink needs to be a fluid sufficient to facilitate extrusion through the printer nozzle, and the printed structure needs to remain undamaged over time at physiological pH.

[0004] Examples of materials used for inks for biological applications are alginate, fibrin, and collagen.

[0005] Collagen is one of the most abundant proteins in nature and is involved in maintaining the structural integrity of tissues. Since collagen is the most abundant component in the extracellular matrix, it is the most used material in cell applications, and this material is used, for example, as a support matrix to assist cell growth. Collagen fibers have a specific amino acid sequence, which enables cell adhesion and rapid growth. The collagen matrix is very useful and has produced several important biological advances. However, an optimal 100% collagen matrix has not been developed. Compositions consisting only of collagen are usually made from soluble collagen and do not usually have the consistency and hardness sufficient to be used as an ink that maintains the integrity of the printed structure without the aid of a cross-linking agent, nor do they have a natural structure.

[0006] In the case of collagen, it can be provided in the form of a hydrogel to have sufficient viscosity and stiffness for use as an ink. An example of this can be found in the patent publication no. CN106581753, which describes a biological hydrogel for 3D printing of a matrix for the proliferation of dermal tissue. 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 an ink, but the structures obtained thereby lose their integrity if the crosslinking agent is not added to the collagen so that they can perform their function.

[0007] Strategies have been employed to functionalize collagen in order to impart the necessary characteristics for use as a 3D printing ink in biological applications. Patent publication number CN106237383 is an example, describing collagen microfilaments functionalized with growth factors that promote cell proliferation and differentiation. In this case, fiber classification and pre-selection are also performed to obtain very small-sized fibers, adjusted so that the size is smaller than the diameter of the extrusion head needle (e.g., a 27G needle has an inner diameter of 210 μm), which are based on soluble collagen extracted with surfactants, such as SDS and triton, and further modified with other molecules to obtain optimal results. Furthermore, the rheological data or hardness achieved by the ink, or the printability or integrity of the resulting structures, are not shown. In the aforementioned patent, fibers ranging from 100, 75, 38, and 25 μm are selected.

[0008] Another strategy used for manufacturing collagen matrices for 3D printing is cryogenic printing, which involves depositing a low-viscosity collagen solution at sub-zero temperatures and performing crosslinking in situ.

[0009] The extrusion and purification methods currently applied to process collagen significantly reduce its crosslinking density, resulting in effects on its related properties and biological responses. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] China Patent Publication No. CN106581753 [Patent Document 2] China Patent Publication No. CN106237383 [Overview of the project] [Problems that the invention aims to solve]

[0011] Therefore, based on the current state of the technology, it is necessary to develop a fibrous collagen-based ink that does not denature and has sufficient consistency and hardness to be used as an ink in 3D printing without the aid of crosslinking agents. [Means for solving the problem]

[0012] Collagen is the most abundant protein in the extracellular matrix of mammalian connective tissues, such as skin, bone, cartilage, and tendons, making up over 90% of their dry mass.

[0013] The essential unit of collagen consists of three polypeptide chains that form an intertwined triple helix, forming a high-molecular-weight unit called tropocollagen. Tropocollagen molecules come together to form collagen fibers.

[0014] In this invention, a collagen ink for 3D printing has been developed that has a viscosity suitable for use in 3D printers and maintains the collagen in its natural state. The ink of this invention is characterized by being a dispersion of natural collagen fibers in an acidic aqueous medium.

[0015] The acidic medium causes the collagen to absorb water, resulting in a fluid that can be extruded.

[0016] Accordingly, a first aspect of the present invention relates to a 3D printing collagen ink comprising a dispersion of natural collagen fibers in an acidic medium having a mass concentration between 0.1% and 10% and a pH between 0.5 and 5, wherein the dispersion has a viscosity between 10 centipoise (cP) and 50 million centipoise (McP) (Brookfield method at a temperature between 18°C ​​and 22°C), and the fiber dispersion comprises fibers having a length in the range of 1 μm to 2500 μm and a diameter in the range of 0.1 μm to 180 μm, as measured at a pH between 1 and 2.

[0017] The fibers in the ink of the present invention have a wide range of lengths and diameters, and sorting is not required to obtain and select small-sized fibers for the ink. The ink is printable even when large fibers, which may be longer than 1 mm and have a diameter greater than the inner diameter of the printer's extrusion head, for example, 22G = 152 μm, constitute a large proportion of its mass.

[0018] The collagen fibers of the present invention maintain a triple helix molecular structure without denaturation. The fibers can absorb water and swell, and their diameter is mainly between 5 μm and 35 μm, although they would not exist if they were denatured or hydrolyzed.

[0019] As mentioned, collagen molecules are elongated molecules composed of three intertwined polypeptide chains, forming a triple helix stabilized by hydrogen bridging bonds or interactions, which is the structure of natural collagen. When collagen is denatured, for example by heating in the presence of water or to an extreme pH value, these chains separate and dissolve to form gelatin. Gelatin formation is due to the breaking of hydrogen bonds that stabilize the collagen triple helix, and the process of converting collagen to gelatin is considered a typical denaturation process. In the collagen fiber dispersion of the present invention, gelatin is either absent or its presence is not significant.

[0020] In this specification, "native collagen" is understood to mean collagen that is not completely or significantly denatured, hydrolyzed, or gelatinized.

[0021] The ink of the present invention is provided in the form of a dispersion of optimally sized natural fibrous collagen in an acidic medium, derived from the biological origin of collagen, and is typically composed of large bundles and / or fibrous aggregates subjected to a process that causes decomposition into smaller diameter and length fibers, allowing for the design of a fluid with rheological properties suitable for precise 3D printing while maintaining its natural structure.

[0022] Structures obtained using the ink of the present invention by 3D printing are stable and durable structures made of natural, insoluble fibrous collagen, without the need for crosslinking agents.

[0023] It is important that the ink contains natural collagen, because in its natural environment, collagen is not only a structural and supportive protein, but also, in particular, interacts with other biomolecules and mediates cell adhesion, thereby guiding cellular function. The ink of the present invention, insofar as it retains its natural structure, will be able to maintain its physiologically relevant characteristics when used for printing in biomedical applications.

[0024] Similarly, the present invention relates to any hydrogel containing the ink described in the first aspect of the present invention and the embodiments described below.

[0025] A second aspect of the present invention is a method for obtaining a collagen ink, the steps being: a) washing and mincing a collagen-containing tissue; b) chemically macerating the minced tissue; c) washing the product obtained in step b) with water; d) adjusting the pH of the by-product obtained in c) to a value between 0.5 and 5 and swelling the by-product of step c); e) mechanically milling the product of step d); f) dispersing in water to a mass concentration preferably between 0.1% and 10% and including.

[0026] The collagen matrix has low immunogenicity, excellent biocompatibility and biodegradability, and in particular contains specific sequences that interact with other biomolecules and mediate the control of cell morphology, adhesion, migration and differentiation. The ink of the present invention has a viscosity suitable for passing through a 3D printing nozzle.

[0027] Therefore, a third aspect of the present invention relates to the use of the ink of the present invention in 3D printing. The present invention also describes a method of printing using the inks described above and their embodiments.

[0028] A fourth aspect of the present invention relates to a structure containing the ink of the present invention. One advantage of this structure is that it is highly biocompatible and absorbent.

Brief Description of the Drawings

[0029] [Figure 1] Figure 1 shows a mesh printed with the ink of the present invention as a biodegradable scaffold for cell culture. [Figure 2]Figure 2 shows a mesh culture containing mouse germinal cell fibroblasts obtained on day 2 of culture, in the same mesh as in Figure 1 (10x). [Figure 3] Figure 3 shows the culture on day 5. [Figure 4] Figure 4 shows the culture after 6 days of growth. [Figure 5] Figure 5 shows the stress sweep plots for samples 531-010 and 531-020. [Figure 6] Figure 6 shows the stress sweep plots for samples 431-010 and 431-020. [Figure 7] Figure 7 shows a structure printed with the ink of the present invention. [Figure 8A] Figure 8A shows various structures made using ink. [Figure 8B] Figure 8B shows various structures created with ink. Figure 8B, 431-010 corresponds to structures A, C, and E after neutralization and adjustment to physiological pH (pH 7.4). [Figure 8C] Figure 8C shows various structures made using ink. [Figure 8D] Figure 8D shows various structures made using ink. [Figure 8E] Figure 8E shows various structures made using ink. [Figure 8F] Figure 8F shows various structures created with ink. Figure 8F corresponds to structures A, C, and E after neutralization and adjustment to a physiological pH (pH 7.4). [Figure 9] Figure 9 shows optical microscope images of individual particles of ink 531-010 of the present invention, diluted to 0.1% and stained with Sirius Red. [Figure 9B] Figure 9B shows optical microscope images of individual particles of ink 531-010 of the present invention, diluted to 0.1% and stained with Sirius Red. [Figure 10]Figure 10 shows the components printed with the ink of Example 8. [Modes for carrying out the invention]

[0030] As described 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 having a concentration between 0.1% and 10% and a pH between 0.5 and 5, wherein the dispersion has a viscosity between 10 cP and 50 McP (according to the Brookfield method at a temperature between 18°C ​​and 22°C as described in Example 3), and the fiber dispersion comprises fibers having a length in the range of 1 μm to 2500 μm and a diameter in the range of 0.1 μm to 180 μm when measured at a pH between 1 and 2.

[0031] Preferably, 90% of the collagen fiber masses in the dispersion consist of fibers with a length in the range of 50 μm to 2500 μm.

[0032] Preferably, 75% of the collagen fiber clumps in the dispersion consist of fibers with a length in the range of 50 μm to 1000 μm, and 50% consist of fibers between 100 μm and 500 μm.

[0033] Preferably, 80% of the collagen fiber mass consists of fibers with a diameter between 5 μm and 35 μm.

[0034] Preferably, the concentration is between 1% and 5%.

[0035] Preferably, the pH is between pH 1 and 3.

[0036] Preferably, the viscosity is between 2 McP and 15 McP.

[0037] A key feature of the collagen matrix of this invention is its integrity after being neutralized to physiological pH.

[0038] A key feature of the collagen matrix of the present invention is its integrity after neutralization to physiological pH. Among the possible applications of the ink of the present invention is the printing of matrices for cell and tissue cultures. In this case, it is highly advantageous that the collagen is natural, as this method closely resembles the original structure of collagen and very similarly reproduces the original environment in which cell proliferation occurs in the body. Remarkably, the resulting structure or matrix is ​​rigid enough to remain undamaged and firm over time at physiological pH. Preferably, the ink used in these applications comprises a dispersion of natural collagen fibers in an acidic medium having a pH between 1 and 3 at a concentration between 2% and 5%.

[0039] The preferred use of the ink described in the previous paragraphs is in matrix structures for cell and tissue cultures.

[0040] As stated above, a second aspect of the present invention is a method for obtaining the ink of the present invention from collagen-containing tissue, comprising the steps: a) A process of washing and cutting collagen-containing tissue, b) A process of chemically macerating the shredded tissue, c) A step of washing the product obtained in step b) with water, d) The pH of the by-product obtained in step c) is adjusted to a value between 0.5 and 5, and the by-product of step c) is swollen. e) A process of mechanically engraving the product of step d), f) A step of dispersing in water at a concentration preferably between 0.1% and 10%. This includes methods.

[0041] Preferably, the collagen-containing tissue is connective tissue. The connective tissue is preferably selected from the dermis, bone, tendon, cartilage, and intestine. Preferably, the connective tissue is dermal tissue, and more preferably, the collagen is extracted from a layer called corium.

[0042] The connective tissue can originate from any animal source. In certain embodiments, the connective tissue may be derived from cattle, sheep, pigs, birds, fish, or a mixture thereof. Preferably, the connective tissue is of cattle origin and from animals older than one year, preferably between one and three years.

[0043] More preferably, if the connective tissue is the dermis, it is depilated and bleached before step a). A bleaching step with a weak oxidizing agent is preferably performed in addition to the washing step a). An example of a weak oxidizing agent is diluted hydrogen peroxide.

[0044] Step b) is preferably carried out in the presence or absence of an enzyme, in the presence or absence of an alkaline agent, such as calcium hydroxide or sodium hydroxide. In particular, step b) is carried out with a sulfur salt and an alkaline treatment agent, such as Na2S and Ca(OH)2.

[0045] Preferably, the acid used for acidification in step d) is selected from hydrochloric acid, lactic acid, acetic acid, and citric acid. Preferably, the pH is acidified to between 1 and 3.

[0046] Preferably, step f) of preparing an aqueous dispersion of fibrous collagen is carried out at a concentration between 1% and 5%. More preferably, it is between 2% and 5%.

[0047] In particular, it is preferable to subject the dispersion obtained in step f) to a mechanical homogenization process, which involves subjecting the dispersion to shear force by passing it through a slit at high pressure, preferably a minimum pressure of 50 atmospheres. Preferably, the pressure is between 50 atm and 100 atm.

[0048] Example 3 and Figures 5 and 6 demonstrate that the homogenization process causes a rheological change in the ink of the present invention. The homogenized mass can withstand greater forces without losing its structure. The elastic modulus (G') value of the collagen mass remains constant even when subjected to greater deformable forces than the unhomogenized mass.

[0049] The present invention also relates to the methods of the present invention described in the preceding paragraphs and the products obtained by all embodiments thereof.

[0050] The printing method using the ink of the present invention has various embodiments.

[0051] In one embodiment of the printing method, the ink is mixed with cells selected from, in particular, 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.

[0052] In another embodiment of the printing method, before printing, the ink is preferably neutralized to a pH between 7 and 8, in particular with a salt or buffer selected from phosphate-buffered saline (1X and 10X PBS), Tricin, MOPS (3-(n-morpholino)propanesulfonic acid), HEPES (4-2(hydroxyethyl)piperazine-1-ylethanesulfonic acid), Tris, and sodium carbonate. [Examples]

[0053] The following examples are merely illustrative of the present invention and should not be construed as limiting the invention.

[0054] (Example 1) To prepare the ink of the present invention, the skin of bovine animals aged 18 to 26 months was used, which had been subjected to a standard depilation process (for example, using a sulfur salt such as Na2S).

[0055] Next, the skin is separated, and the dermis, from which collagen is obtained, is extracted.

[0056] After washing several times with water and bleaching with a weak oxidizing agent (e.g., diluted hydrogen peroxide), the material is filled with calcium hydroxide under the time and temperature conditions necessary for its sufficient maceration. The remaining reagent is then washed again with water and acidified with concentrated HCl (33%). It is chopped using a grinder and diluted with water until a 2.9% aqueous collagen dispersion is obtained. The resulting pH is 3.1.

[0057] (Example 2) The dispersion and mesh-type matrix (5 x 2.5 cm and 1.2 mm pore size) from Example 1 were 3D printed in a 10 cm Petri dish using a 24 G nozzle needle extrusion head at a speed of 3 mm / s to serve as scaffolds for cell culture substrates.

[0058] After printing the mesh, it was covered with 50 mM NaOH to raise the pH and thereby coagulate the collagen. After coagulation, the mesh was covered with a sufficient amount of pH 7.4 phosphate buffer (PBS) and subsequently immersed in an antibiotic-fortified culture medium. It should be noted that this dispersion of the present invention was printed and coagulated at room temperature without the addition of a crosslinking agent.

[0059] Mouse germinal fibroblasts were cultured on a mesh. A total of 300,000 cells were resuspended.

[0060] Figure 2 shows a mesh culture containing fibroblasts obtained after 2 days. Figure 3 shows the culture on day 5. The culture dish is 100% confluence, and tactism and directional cell proliferation due to the material begin to be observed. Figure 4 shows the culture on day 6, showing proliferation. Some cells are growing in the direction of the collagen fibers in the mesh.

[0061] (Example 3) Rheology of Ink For this purpose, a Modulate Advanced Rheometer System (MARS 40) from Thermo Haake, equipped with a 20 mm plate-plate rotor, was used. Vibration stress sweep tests were performed with a stress range (τ) of 1 Pa to 15,000 Pa, a fixed frequency of 1 Hz, and a measurement temperature of 15.0 °C, maintaining a plate spacing of 0.8 mm. Before the test, once the 0.8 mm spacing was reached, the samples were tempered between the plates for 10 minutes to ensure the measurement temperature.

[0062] By performing stress sweeps at a fixed frequency, the linear viscoelastic region can be identified.

[0063] Since the reaction of a material depends solely on its structure and not on rheological parameters such as stress or deformation, vibration measurements in the linear viscoelastic region are extremely important.

[0064] During the test, the values ​​of G' and G'' (vertical axis) are shown as functions of stress (horizontal axis). The values ​​of the intersection, LVR, and yield value (flow limit) correspond to stress, i.e., the horizontal axis.

[0065] The modulus of elasticity or storage modulus (G') relates to the energy stored in the material, while the viscous modulus or loss modulus (G'') relates to the energy dissipated by the material.

[0066] The intersection is the point where G' and G'' are equal. It coincides with the value of the vibrational shear stress at which the curves of G' and G'' intersect, and therefore, it is the stress value at which the mass stops behaving as an elastic material and begins to behave as a viscous fluid.

[0067] The intersection value of the homogenized mass is approximately 40% higher than that of the diluted mass, meaning that the mass maintains its elastic behavior under higher stress and can therefore withstand greater stress without losing its structure.

[0068] The yield point of a material can be determined by stress sweeping. While the material is not flowing (solid behavior), the value of the elastic modulus remains constant, but when the material begins to flow, its value decreases. The value of the elastic modulus at which the material begins to flow is the yield value (flow limit), which indicates the point at which the mass begins to undergo permanent deformation. Up to that point, the deformation suffered by the mass is recoverable, and therefore the values ​​of G' and G'' remain constant. However, from the yield value (flow limit) onward, the mass begins to lose its elastic component, and the viscous component increases until they become equal at the intersection and the mass begins to behave like a viscous fluid. Figures 5 and 6 show stress sweep graphs for samples 531-010, 531-020 and 431-010, 431-020.

[0069] Samples 531-010 and 431-010 are equivalent to those in 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 dispersion, but have been subjected to a high-pressure homogenization process in an industrial homogenizer, which basically involves passing the mass through a slit at a pressure between 50 and 300 atmospheres using a piston.

[0070] The diluted and homogenized lumps of 531-020 and 431-020 have a higher yield value (flow limit) than the undiluted and unhomogenized lumps of 531-010 and 431-010, and can therefore be subjected to greater stress while maintaining their elastic components without damage and ultimately preserving their structural properties.

[0071] [Table 1]

[0072] A rapid method for quantifying the degree of degradation and hydrolysis of natural collagen involves determining the soluble fraction in 10% ammonium sulfate after centrifugation at 18,000 rpm. Subsequently, the collagen protein content of the fraction is quantified using the biuret method, and the result is expressed as a percentage relative to the percentage of collagen previously quantified using the biuret method. This fraction is particularly evident in degraded collagen.

[0073] Next, the collagen protein content of the aforementioned fraction is quantified using the Biuret method, and the results are expressed as a percentage (Table 2).

[0074] [Table 2]

[0075] The viscosity of the dispersion of the present invention was measured.

[0076] The viscosity of the ink of this invention was determined using a Brookfield viscometer model DV2T HBT (Brookfield Ametek). The viscosity was calculated from the measured torque based on the selected spindle and rotational speed.

[0077] The spindle used in this method is an inverted T-type (Helipath with T-Bar Spindle). The following are the viscosity ranges measured by each spindle of the viscometer model.

[0078] [Table 3]

[0079] For lumps with a collagen concentration of 2% or less, and in the lower viscosity range, use a different spindle.

[0080] [Table 4]

[0081] The apparatus requires specifying the spindle and rotational speed to be used, and is configured to perform the conversion of cP to viscosity itself.

[0082] For this purpose, the mass was placed in a 250 ml beaker. The temperature of the mass was within the range of 18°C ​​to 22°C. Depending on the predicted viscosity of the mass, the corresponding spindle was used. Therefore, spindle 94 was used for the homogenized mass (531-020), while spindle 95 was used for the non-homogenized mass (531-010). The rotation speed was 1.0 RPM, and 250 measurements were taken over 4 minutes.

[0083] Viscosity measurements showed a significant reduction after homogenization, reaching 23.3 and 6.5 McP for 531-010 and 531-020, respectively (Table 1). This reduction in viscosity greatly improves the printability of the ink and reduces the pressure required for printing by the printer.

[0084] (Example 4) Effect of pH on ink In this case, the behavior of the ink of the present invention and the behavior of the neutralized ink of the present invention were compared. It is important to maintain the structure and hardness of the material after neutralization to physiological pH, and to ensure that the structure and shape of the printed 3D structures remain sufficiently hard and stable under the physiological conditions required for use in biomedical applications where they are used as supports for cell proliferation. As shown in the following table (Table 3 (Table 5)), although the elastic modulus is reduced, the G' values ​​of the neutralized materials (531-010 neutral and 531-020 neutral) remain high. Therefore, the materials remain sufficiently hard over time after being neutralized to physiological pH with various bases and placed in a medium similar to cell culture medium, such as PBS, calcium-fortified Hanks solution (HBSS-Ca), or equivalent.

[0085] Rheological analysis was performed using frequency sweeps consisting of vibration tests with a fixed stress of 100 Pa and a measurement temperature of 15°C, with a plate spacing of 0.8 mm in the range of 0.01 Hz to 100 Hz. The table below shows the values ​​obtained for a frequency of 0.1 Hz. After neutralizing to physiological pH with NaOH (0.05 M) and equilibrating in PBS, the stability of the material of the present invention in Example 3 at a collagen concentration of 4.8% can be observed, and it maintains a very high modulus of elasticity (G').

[0086] [Table 5]

[0087] Similarly, the time-dependent stability of the neutralized material was determined. For this purpose, another batch (batch 6) prepared as the gel of the present invention in 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 covered with the neutralizing agent.

[0088] The table below shows that, after neutralization with NaOH, washing, and equilibration with PBS at physiological pH, the material maintains a high and stable modulus of elasticity over time, although it initially decreases. This structural maintenance is confirmed under physiological conditions equivalent to those used in cell culture (37°C and pH 7.4). Similar results are observed with other bases, such as ammonium hydroxide, and other equilibration media, such as HBSS-Ca, confirming the maintenance of stable structural shapes in normal culture media.

[0089] [Table 6]

[0090] (Example 5) Printing with the ink from Example 3 The device pressure was appropriately adjusted (550 kPa), and printing was performed using a 22G nozzle needle extrusion head at an extrusion speed of 2 mm / second. Despite the ink containing insoluble collagen fibers, the continuity of the structure is evident, as shown in Figure 7.

[0091] (Example 6) Printability of the ink of the present invention Figures 8A to 8F show examples of structures obtained with the ink from Example 3 at an extrusion speed of 5 mm / second by appropriately adjusting the pressure of the 3D printer. The continuity of the structure can be seen.

[0092] Figures 8E to 8F represent multi-layered 3D structures.

[0093] Figures 8B, 8D, and 8F show the same structure on the left, neutralized with 50 mM NaOH and adjusted with PBS to a physiological pH of 7.4, which is the pH most favorable for cell proliferation. In all cases, the integrity of the structure is maintained, and it can be manipulated for the applications described above.

[0094] When the ink of the present invention is prepared at a concentration of less than 1%, a significant decrease in the hardness of the structure is observed, and the viscosity value for the 1% collagen ink is 6,000 cP. Therefore, these conditions are not most advantageous when the structure or matrix is ​​neutralized, but the ink may be useful when the printed structure does not require neutralization.

[0095] The same thing happened when the ink's pH was lowered excessively; when the ink's pH was 0.6, the elasticity of the 531-020 ink decreased by reducing the G' value to 3900 Pa and the viscosity to 3.14 McP. After printing, the structure lost its hardness during the neutralization process.

[0096] The use of hydrochloric acid, lactic acid, or acetic acid during the acidification process in step d) of the manufacturing process resulted in a fully printable ink that maintained the integrity of the structure after neutralization at the pH and concentration values ​​mentioned above.

[0097] (Example 7) Morphological analysis Morphological analysis of inks 531-010 and 531-020 from Example 3 was performed by video analysis. For this purpose, the inks were diluted to approximately 1% in deionized water and adjusted to approximately 1.5 pH with 1M HCl. The diluted samples were vortexed at 2500 rpm for 10 minutes and then diluted to 1 / 20 with deionized water.

[0098] Particle size measurements were performed using a Sympatec QICPIC / Lixell particle analyzer with a continuous float tray (0.5 mm width) in the particle size measurement range of 4 to 2888 μm. Due to the large heterogeneity of particle distribution typical of collagen suspensions, at least three individual measurements were performed on two secondary samples. In total, between 1 million and 3 million particles were detected per sample.

[0099] The evaluation was based on the distribution of fiber length and fiber diameter. The proportion of fibers of a specific length or width in the entire mass of collagen fibers in the sample was calculated. Tables 5 (Table 7) and 6 (Table 8) show the cumulative distribution of length and diameter (in μm) for 10%, 16%, 50%, 84%, and 90% of the collagen fibers in the entire mass, calculated from the mean values.

[0100] [Table 7]

[0101] [Table 8]

[0102] As shown in Table 5 (Table 7), sample 9D exhibits a broader fiber length distribution. The median is 838 μm. This means that 50% of the total collagen fiber mass in the sample is supplied by fibers longer than 838 μm. 84% of the collagen mass is supplied by fibers longer than 336 μm. However, in 531-020, the proportion of smaller fibers is very high. Its median of 383 μm is considerably lower than that of sample 531-010. This means that half of the collagen mass in sample 531-020 consists of fibers shorter than 383 μm, compared to 838 μm in the case of 531-010.

[0103] Therefore, homogenization reduces the size of the fibers, which facilitates the movement of ink through the extrusion head, ultimately improving its printability and reducing the pressure required to print it on commercially available printers.

[0104] The cumulative distribution of fiber diameters for samples 531-010 and 531-020 (Table 6 (Table 8)) is almost identical, with a median of 19 μm, and 80% of the fibers being in the diameter range of 10 μm to 31 μm.

[0105] Figures 9A and 9B show optical microscope images of individual particles of ink 531-010 of the present invention, diluted to 0.1% and stained with Sirius Red. The figures provide a visual image of the fibers present in the ink of the present invention, and a broad distribution of fiber length and fiber diameter is clearly observed.

[0106] (Example 8) Before printing, the acidic ink from Example 1 was neutralized to a concentration of 5%, and then mixed with 1 M Tris(hydroxymethyl)aminomethane (Tris) adjusted to pH 7.4 in a ratio of 3:2 (V:V) to obtain a concentration of 3%. The ratio could be varied to obtain a more diluted ink. Similarly, half of the buffer could 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 another syringe was filled with 2 ml of Tris. The two syringes were connected using a connector, and the contents were passed from one to the other. This final step was repeated 40 times to uniformly mix the contents. Subsequently, printing was performed using a 20G nozzle needle extrusion head at an extrusion speed of 5 mm / s with a pressure adjusted to 70 kPa. The resulting pattern is shown in Figure 10.

[0107] (Example 9) Cell encapsulation in neutral collagen ink The amounts of cell density proposed in this example are based on studies using mouse fibroblasts. The method for mixing the different components used in this example is the same as that for the neutralized ink in Example 8.

[0108] As described in Example 1, a collagen mass was obtained at a neutral pH. In this case, the amount required for subsequent mixing with cells was calculated from the amount of the collagen mass mixture at an acidic pH with the corresponding buffer. For this purpose, three parts were mixed with the cells: collagen, one of the buffers, and one of the DMEM (Dulbecco's Modification of Eagle's Medium) culture media. In this case, the acidic collagen mass was mixed with the corresponding buffer 40 times (as described in the previous examples of neutralized ink), and then similarly mixed with the culture medium containing cells, but this time a total of 20 times.

[0109] To obtain a culture medium containing cells, a standard cell collection protocol was followed, and the cells were obtained in a suspension with an approximate density of 200,000 cells / ml.

[0110] After mixing all components, the structure was bioprinted onto plates prepared for cell culture. Once the structure was bioprinted, reinforced DMEM was added to cover the entire scaffold or structure. A live / dead assay was performed 5 days after cell bioprinting. To supplement the study, the scaffold (support) was digested using a combined treatment of trypsin-EDTA and collagenase to obtain cells from both the surface and those embedded in the ink (first a 20-minute incubation with 0.05% trypsin-EDTA, followed by a second incubation with 500 U / ml collagenase until the scaffold was completely dissolved). The results showed a cell viability of over 90%.

Claims

1. A collagen ink for 3D printing comprising a dispersion of natural collagen fibers in an acidic medium having a mass concentration between 0.1% and 10% and a pH between 0.1 and 5, It has a viscosity between 2 McP and 15 McP, as measured by the Brookfield method at temperatures between 18°C ​​and 22°C. At least 75% of the collagen fiber mass in the dispersion contains fibers whose length, as measured at a pH between 1 and 2, is in the range of 50 μm to 1000 μm. 80% of the collagen fiber mass contains fibers with a diameter between 5 μm and 35 μm, measured at a pH between 1 and 2. Collagen ink for 3D printing.

2. The 3D printing collagen ink according to claim 1, characterized in that 50% of the collagen fiber clumps in the dispersion contain fibers having a length in the range of 100 μm to 500 μm.

3. The 3D printing collagen ink according to claim 1 or 2, characterized in that the mass concentration is between 1% and 5%.

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

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

6. A method for obtaining an ink according to any one of claims 1 to 4 from collagen-containing tissue, comprising the steps: a) The process of washing and cutting collagen-containing tissue; b) A step of chemically macerating the cleaved tissue in the presence of calcium hydroxide, sodium hydroxide, or a sulfur salt and an alkaline agent; c) A step of washing the product obtained in step b) with water to obtain a by-product; d) A step to adjust the pH of the by-product obtained in step c) to a value between 0.5 and 5, to swell the by-product of step c), and to obtain the product; e) A process of mechanically engraving the product of process d); f) A process of dispersing in water to a mass concentration between 0.1% and 10%. Methods that include...

7. The method according to claim 6, characterized in that the collagen-containing tissue is connective tissue.

8. The method according to claim 7, characterized in that the connective tissue is dermis tissue of a layer called corium.

9. The method according to claim 7, characterized in that the connective tissue is derived from a bovine between 1 and 3 years of age.

10. The method according to any one of claims 6 to 9, characterized in that step c) is carried out in the presence of an alkaline agent.

11. The method according to any one of claims 6 to 10, characterized in that in step d), the pH is acidified to a pH between 1 and 3.

12. The method according to any one of claims 6 to 11, characterized in that a step of homogenizing the product obtained in step d) is included before step e).

13. The method according to any one of claims 6 to 12, characterized in that step f) is performed at a mass concentration between 1% and 5%.

14. Use of the ink according to any one of claims 1 to 4 in printing a 3D structure.

15. A structure containing the ink described in any one of claims 1 to 4.

16. A method for printing using the ink described in any one of claims 1 to 4, characterized by comprising the step of mixing the ink described in any one of claims 1 to 4 with cells before printing.

17. The method according to claim 16, characterized in that the cells are 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.

18. A method for printing ink according to any one of claims 1 to 4, comprising the steps: The process involves neutralizing the aforementioned ink to a physiological pH of 7 to 8, A method characterized by including a step of printing a structure.

19. The method according to claim 18, characterized in that the ink is mixed with cells.

20. The method according to claim 18, characterized in that the neutralization is carried out with a salt or buffer selected from phosphate-buffered saline (PBS 1X and 10X), Tricinene, MOPS, HEPES, Tris, and sodium carbonate.

Images