Bioink and scaffold for corneal tissue regeneration and method of manufacture

Abstract

The invention relates to a bioink for 3D printing of a scaffold (1) suitable for corneal tissue regeneration, composed of sodium alginate, aldehyde hyaluronic acid, N,O-carboxymethyl chitosan and collagen I, where the collagen I undergoes a preliminary neutralisation process using sodium hydroxide. The present invention also describes a method for the 3D printing of a scaffold (1) suitable for corneal tissue regeneration and a method for sterilising same using ethylene oxide (EtO).

Description

[0001] DESCRIPTION

[0002] Bio-ink and scaffold for corneal tissue regeneration and manufacturing method

[0003] Technical Field

[0004] The present invention belongs to the field of biomedicine, more specifically to the design and development of bio-inks and scaffolds for corneal tissue regeneration.

[0005] Background of the invention

[0006] The cornea plays a crucial role in visual function by acting as the primary refractive lens of the human eye. Due to its exposure to the external environment, the cornea is subject to various conditions, such as trauma, infections, and degenerative diseases, which can compromise its transparency and structure, causing significant vision loss. In many cases, corneal transplantation has been the traditional therapeutic solution; however, the shortage of donors, the risk of immune rejection, and postoperative complications limit its effectiveness and availability.

[0007] Faced with these challenges, tissue engineering has emerged as a promising strategy for corneal tissue regeneration. Within this discipline, the development of bioinks and three-dimensional scaffolds has become particularly relevant. Bioinks, composed of natural, synthetic, or hybrid polymers in combination with corneal cells, offer a solution for 3D printing customized structures that mimic the mechanical and optical properties of native corneal tissue. Among the known solutions are bioinks based on collagen, methacled gelatin (GelMA), and other photocurable hydrogels that allow for the encapsulation of living cells while maintaining adequate optical properties. However, many of these bioinks have significant limitations, such as low mechanical stability, difficulties in long-term transparency retention, and a limited capacity to support cell proliferation.

[0008] Meanwhile, the scaffolds used in corneal regeneration are manufactured using various techniques, such as electrospinning and 3D printing. Materials like polylactic acid (PLA) and aliphatic polyester have been used to provide structural support to the regenerated tissue. However, these materials are often opaque or rigid, which hinders their integration and functionality as corneal substitutes.

[0009] In recent years, various solutions have been developed to address these challenges. Among them, for example, patent ES2667821B1 describes bioartificial membranes with controlled stiffness and viscoelasticity, intended for applications in tissue engineering, including corneal regeneration. These membranes aim to mimic the mechanical properties of the cornea, improving the biocompatibility and functionality of the implant. However, this solution does not guarantee long-term optical transparency, as viscoelasticity can decrease structural stability and generate opacities as the material degrades. Furthermore, its ability to support specific cellular integration of corneal tissue is not yet fully validated.

[0010] Patent ES2710024T3 describes a collagen biotissue and its use in surgical grafts, including ophthalmic applications. This biotissue offers a structure compatible with corneal tissue, although it can become opaque due to collagen retraction or uneven water absorption. Its mechanical functionality is also limited, especially in the dynamic environment of the eye, making prolonged use without loss of optical properties difficult.

[0011] Patent ES2200001T3 presents a biocompatible, optically transparent, collagen-based polymer material designed for ophthalmic applications such as contact lenses or corneal implants. While this patent offers transparent materials, the collagen-based polymer composition has limitations in terms of mechanical stability and resistance to degradation in ophthalmic environments. Furthermore, the material may lack the necessary elasticity to fully integrate with the corneal curvature and allow for adequate cell proliferation.

[0012] Another example is application W02020028268A1, which describes implantable scaffolds and methods for their manufacture using 3D bioprinting. The scaffolds are designed to be biocompatible and functional, enabling the regeneration of specific tissues, including potential applications in the cornea. However, this document does not specify materials that simultaneously guarantee transparency and adequate optical interaction with light. Biocompatibility is limited to general applications, and complete integration into a specialized tissue such as the cornea is not assured.

[0013] As a solution somewhat closer to the present invention, patent KR102430470B1 describes a method for manufacturing transparent corneal tissue using 3D extrusion printing technology. This development focuses on reproducing the organized and aligned structure of collagen to attempt to replicate the transparency and functionality of corneal tissue. However, it presents drawbacks from a biocompatibility standpoint, since the described decelling process does not guarantee complete elimination of immunogenic components, which could generate adverse immune responses. Furthermore, in terms of functionality, the excessive secretion of type I collagen observed after 28 days could alter the scaffold structure and affect its mechanical integrity.

[0014] Despite the advances described, challenges remain in the creation of bio-inks and scaffolds that optimally combine the transparency, functionality, and biocompatibility necessary for effective corneal tissue regeneration.

[0015] Summary

[0016] To overcome the technical problems raised, the present invention proposes a scaffold suitable for corneal tissue regeneration, which is manufactured by 3D printing from a bio-ink composed of: bovine collagen I (COL I) neutralized with NaOH; sodium alginate (ALG); hyaluronic aldehyde (AHA); and N,O-carboxymethylated chitosan (CMC), in the proportions indicated below.

[0017] The scaffold that is the subject of the invention is characterized by being biocompatible, resistant, functional and biodegradable, which allows its integration into the human body without causing adverse reactions.

[0018] The main technical advantages of the scaffolding that is the subject of the invention are:

[0019] - Strength and consistency to allow suturing;

[0020] - stability and homogeneity of the ink printed using 3D technology, which allows it to be customized according to the individual needs of the patient;

[0021] - Transparency, achieving refractive and light transmittance values ​​very similar to those of the human cornea, guaranteeing clear vision during treatment; - Reticular structure with controlled porosity to maintain optical transparency; - Homogeneous arrangement of fibers and improvement of the biological properties of collagen, thanks to the neutralization process, as will be explained later; - Differentiation of mesenchymal cells into keratinocytes, maintaining their structure without degradation for at least 21 days in vitro, allowing sufficient time for tissue regeneration.

[0022] A second aspect of the present invention consists of the 3D printing method of a scaffold suitable for corneal tissue regeneration.

[0023] Brief description of the figures

[0024] Figure 1 shows the 3D design of the scaffold of the invention. In one embodiment, the scaffold has a diameter of 11 mm and a height of 0.5 mm.

[0025] Figure 2 shows the closed syringe system used to mix the collagen with the other components of the bioink of the invention.

[0026] Figures 3A and 3B show, respectively, the percentage transmittance in the visible light spectrum and the refractive index of the four materials analyzed: the acid ink (INK 5), the neutralized ink of the invention (INK 5_NEU), the acid scaffold (S5), and the neutralized scaffold of the invention (S5N), compared to the "Control," which is the value of the human cornea. The measurements in Figure 3B were performed using an ABBE WAY-2S refractometer at 37°C.

[0027] Figure 3C is an image in which the transparency of the neutralized scaffold of the invention (S5N) can be visually observed.

[0028] Figure 3D shows the tear strength index of the neutralized scaffold of the invention (S5N) versus the acid scaffold (S5).

[0029] Figure 3E shows the mechanical strength index of the neutralized scaffold of the invention (S5N) versus the acid scaffold (S5), according to Young's modulus.

[0030] Figure 3F shows the mechanical strength index of the neutralized scaffold of the invention (S5N) compared to the acid scaffold (S5), according to the Compression modulus. Figure 4A shows the swelling percentage of the neutralized scaffold of the invention (S5N) compared to the acid scaffold (S5).

[0031] Figure 4B shows the mass loss or acellular degradation of the neutralized scaffold of the invention (S5N) versus the acid scaffold (S5).

[0032] Figure 4C shows SEM images of the surface of the neutralized scaffold of the invention (S5N) versus that of the acid scaffold (S5).

[0033] Figures 5A and 5B show the results of the biocompatibility tests of the neutralized scaffold of the invention (S5N) against the acidic scaffold (S5).

[0034] Figures 6, 7 and 8 show the immunofluorescence results of the neutralized scaffold of the invention (S5N) against the acidic scaffold (S5) with respect to Control. Figure 9A shows a graph of the cell viability assay of the neutralized scaffold of the invention (S5N).

[0035] Figure 9B shows a light transmittance graph of the neutralized scaffold of the invention (S5N).

[0036] Figure 10 shows a photograph of the freeze-dried scaffold of the invention (S5N).

[0037] Figure 11 shows: A) Direct and indirect biocompatibility testing. B) Transparency testing of collagen-based scaffolds. Transmittance (T%) values ​​in the visible spectrum (300-700 nm). C) Photograph of the rehydrated scaffold of the invention (S5N).

[0038] Finally, Figure 12 shows the biocompatibility indices of the scaffold of the invention (S5N) cultured with corneal cells and rehydrated. A) Microscope photographs of the scaffold stained using the live-dead assay. The left photograph shows the bright-field image, the middle one the live cells, and the right one the dead cells. B) Percentage absorbance of the CCK8 assay. C) Photographs of the cell scaffold.

[0039] Detailed description

[0040] Biotint and hydrogel

[0041] As detailed below in Table 1, the scaffold (1) of the present invention is manufactured from a bio-ink (INK 5_NEU) composed of sodium alginate (3.5% w / v), hyaluronic aldehyde (0.5% w / v), N,O-carboxymethylated chitosan (0.1875% w / v) and bovine type I collagen (0.135% w / v), the latter neutralized with sodium hydroxide (20 pl / ml).

[0042] Table 1: Polymer concentration (% w / v)

[0043] >

[0044]

[0045] ALG: Sodium Alginate; COL I: Soluble Type I Collagen of Bovine Origin; AHA: Hyaluronic Aldehyde; CMC: N,O-Carboxymethylated Chitosan

[0046] Scaffold manufacturing procedure

[0047] 1. Pretreatment

[0048] Prior to this, the bio-ink of the invention (INK 5_NEU) is subjected to the following treatment in order to obtain the hydrogel finally used to carry out the 3D printing of the scaffold (1):

[0049] The components of the bio-ink (INK 5_NEU), with the exception of collagen, are individually dissolved in phosphate-buffered saline solution without calcium or magnesium. The absence of these ions prevents premature gelation or aggregation, especially in sensitive systems like alginate, which could form a gel in the presence of calcium. Initially, sodium alginate (ALG) is combined with hyaluronic acid (AHA) and N,O-carboxymethylated chitosan (CMC). These last two components form Schiff bases, a type of covalent bond that stabilizes the three-dimensional structure and is favored by a neutral or slightly acidic environment. This mixture is cooled to 4°C. Simultaneously, the collagen is neutralized using a 1M NaOH solution, also at 4°C. Mixing at this temperature offers several advantages.At 4°C, collagen retains its native structure, the triple helix, which is responsible for its mechanical strength and biological properties. Furthermore, at this temperature, it does not begin to form gel networks, ensuring that the material remains in a liquid state. This facilitates neutralization and handling before use, resulting in a homogeneous solution and a hydrogel with improved properties. Additionally, collagen neutralization reactions generate localized heat, and performing the process at a low temperature helps dissipate this heat, preventing damage to the collagen. Finally, working at this temperature does not replace sterility, but it does reduce the proliferation of microorganisms.

[0050] The resulting solution is combined using a closed syringe system. As shown in Figure 1, the two syringes are joined by a sterile connector, and the mixture is manually homogenized. In the same syringe, the material is heated to 37°C for 15 minutes to allow the hydrogel to fully polymerize. At this temperature, the collagen self-assembles into organized fibrils, forming a three-dimensional gel network. The neutralized collagen, along with the other components, forms a hydrogel with optimal properties for cell growth. This hydrogel also resists cell growth while maintaining adequate transparency, thus overcoming the main drawbacks mentioned earlier.

[0051] 2. 3D Printing

[0052] The printing was performed using an extrusion 3D bioprinter (BIO-X™, CELLINK). The bio-ink was loaded into a 30 cc UV-protected cartridge equipped with a piston and a 25 G (0.25”) blunt stylus (CELLINK). The 3D models were created in STL format using Blender software, while the G-code was generated with Repetier Host. The bio-ink printing parameters were 10 kPa pressure, 30°C temperature, and a speed of 1 mm / s. A 10 mm diameter, 0.5 mm high cylinder was used for scaffold characterization.

[0053] 3. Post-processing

[0054] The scaffold is built layer by layer, followed by post-processing that includes gelation with calcium chloride, serum washes to remove ionic residues, and lyophilization.

[0055] The resulting membrane has a porous structure and mechanical properties particularly suitable for corneal tissue regeneration. Trials

[0056] The printed membrane has been characterized to determine if its properties are suitable for use in corneal tissue regeneration. Furthermore, its neutralization process has been characterized to gain a deeper understanding of the scaffold's internal structure.

[0057] The acidic ink (INK 5), the neutralized ink of the invention (INK 5_NEU), the acidic scaffold (S5), and the neutralized scaffold (1) of the invention (S5N) were compared. Figures 3A and 3B show the percentage transmittance in the visible light spectrum and the refractive index of these four materials compared to the control, which is the value of the human cornea. The refractive index measurements (3B) were performed using an ABBE WAY-2S refractometer at 37°C.

[0058] Specifically, Figure 3A shows that the transmittance index of the neutralized scaffold (1) of the invention exceeds 90%. Figure 3B shows that the refractive index of the neutralized scaffold (1) of the invention (S5N) is 1.38, very similar to that of the human cornea. Both tests confirm that the neutralized scaffold (1) of the invention (S5N) possesses the appropriate optical properties to allow vision during treatment.

[0059] The transparency of the scaffold (1) of the invention (S5N) is visually observed in Figure 3C, and its tear resistance index compared to the acid scaffold (S5) is shown in Figure 3D. Precipitations formed by collagen are visible on the acid scaffold (S5), indicating that the neutralization process gives the material homogeneity and transparency.

[0060] The mechanical strength of the scaffold is a key factor in the success or failure of the transplant. It must allow surgeons to manipulate and suture the scaffold in the eye, as well as ensure that it maintains adequate strength over time. The structural organization must provide the cornea with specific mechanical properties to withstand an intraocular pressure of 1–3 kPa once implanted, while also facilitating the infiltration and migration of corneal keratocytes into the scaffold to promote corneal regeneration. Scaffold stiffness has been shown to influence cell phenotype, with lower stiffness reducing differentiation into myofibroblasts. Figure 3 shows a compilation of the tear strength (3D), Young's modulus or elastic modulus (3E), and compression modulus (3F) values ​​of the neutralized scaffold (1) of the invention (S5N) versus the acidic scaffold (S5), in relation to their mechanical properties.

[0061] Achieving a Young's modulus similar to that of the human cornea in corneal constructs is essential for obtaining both elasticity and stiffness or strength in the material. The Young's modulus of the cornea exhibits considerable variability in studies, with values ​​ranging from 0.1 to 57 MPa, depending on factors such as the stromal region characterized, the donor's age, storage time, and the measurement methodology used. A recent study in 100 healthy eyes reported an average Young's modulus of 0.29 ± 0.06 MPa. Similar values ​​were obtained when measuring the modulus solely in the stromal layer, both in the anterior and posterior stroma, which ranged from 0.25 ± 0.21 MPa to 0.1 ± 0.06 MPa, respectively. In the measurements of Figure 3F, the acid scaffold (S5) records compression values ​​of 0.012±0.001 MPa, while in the case of the scaffold (1) of the invention (S5N) it is 0.015±0.001 MPa.The Young's modulus values ​​were 0.015 ± 0.002 and 0.012 ± 0.001, respectively, as shown in Figure 3E. These values ​​are consistent with findings from other research on alginate-based hydrogels. It was also observed that both scaffolds withstood an applied deformation of 80% without breaking. The neutralized scaffold (1) of the invention (S5N) withstands greater compressive force; however, it exhibits a lower elastic modulus. These findings indicate that the neutralization process results in a more rigid and durable structure. Finally, the force required to move the thread at a constant speed until the sample broke was measured (Figure 3D). The maximum force was 0.42 ± 0.01 N for the acid scaffold (S5) and 0.53 ± 0.03 N for the scaffold (1) of the invention (S5N). In all samples, this value is significantly higher than the strength required for a surgical suture.It is evident that collagen plays a fundamental role in increasing elasticity, giving the ink a greater capacity to recover its original shape after deformation. It can be concluded that the neutralization process confers rigidity to the bio-ink, but makes the scaffold (1) of the invention (S5N) less elastic.

[0062] To gain a deeper understanding of the scaffolds' microstructure, scanning electron microscopy (SEM) images were correlated with the swelling rate of the polymer mixture. These values ​​provide information about porosity and, consequently, the ability of cells immersed in the ink to diffuse nutrients, signaling molecules, and oxygen.

[0063] The swelling capacity of a hydrogel is intrinsically linked to its crosslinking capacity. Higher density and viscosity are generally correlated with a lower degree of swelling. As shown in Figure 4B, the graph illustrates a percentage increase in mass exceeding 150% in both scaffolds during the first five hours. This increase is expected due to the high water absorption capacity inherent in the biopolymers used in the ink. The high water absorption capacity of biopolymers such as CMC and AHA is fundamentally linked to their hydrophilic nature and the formation of a crosslinked hydrogel network through Schiff-based chemistry, a product of a condensation reaction between the aldehyde group of AHA and the amino group of CMC, which generates a covalent (C=N) bond. This combination allows the resulting material to absorb and retain significant amounts of water.

[0064] Furthermore, microscopy allows for the observation and characterization of materials, providing information on their morphology and chemical composition. The SEM images (120x magnification) in Figure 4C show the surface characteristics of the scaffold, revealing differences between the acidic scaffold (S5) and the neutralized scaffold (1) of the invention (S5N). The latter exhibited a porous structure, in contrast to the acidic sample, possibly due to the way this polymer self-assembles into organized fibrils upon neutralization, resulting in a more open arrangement and the formation of pores. Previous studies have shown that soluble and neutralized COL I can produce similar porous structures. In the field of tissue engineering, one of the main objectives is to optimize the design of porous constructs, improving the biological properties of artificial tissue without compromising mechanical strength.At the microscopic level, it has been established that pores confer advantages in processes such as cell migration and differentiation by facilitating the exchange of nutrients, metabolites, and waste products, thereby promoting the infiltration of host cells into scaffolds after transplantation. The internal morphology of the scaffold (1) of the invention (S5N) revealed a range of pore sizes (between 100 and 1000 pm), which can positively impact corneal regeneration.

[0065] While cell differentiation and multiplication are favored by an optimal microscopic structure, it is also essential to ensure that the material has adequate durability within the organism. Otherwise, it will degrade before cells can grow on it. To this end, these properties are complemented by the study of degradation kinetics. Ideally, degradation should coincide with the regeneration or replacement of native tissue as the scaffold degrades. This test aims to determine the time required for the scaffold to decompose or lose its structural integrity until its eventual failure. Figure 4A shows the acellular degradation rate of both types of scaffolds, incubated at 37°C and in an atmosphere suitable for cell growth. This test concluded that the scaffold (1) of the invention (S5N) remains stable for more than one month in culture medium under cell incubation conditions.In conclusion, it can be stated that the scaffold (1) of the invention (S5N) has an optimal structure and degradability for cell growth.

[0066] Biocompatibility

[0067] Once the neutralization process was optimized and cell-free characterization was completed, the compatibility of the biological components with the inks was evaluated. The direct and indirect tests described in ISO 10993-5 are essential to ensure that materials used in medical devices do not elicit an immune response in the host or cause adverse effects on cells and tissues. Although the cornea is considered an immunoprivileged organ due to the absence of blood and lymphatic vessels, it can still trigger an immune response and undergo rejection, especially in cases of retransplantation. This is primarily due to the initial nerve severing, which leads to the loss of the immune shunt associated with the anterior chamber. A cell viability rate exceeding 95%, as shown in Figure 5, confirms that the material is biocompatible and has no significant cytotoxic effects.No difference in viability was observed between the acidic (INK 5) and neutralized inks of the invention (INK 5_NEU), since the cells are not embedded in the ink; instead, the culture medium provides the appropriate pH to support cell proliferation.

[0068] The researchers also tested whether the scaffolds could support the differentiation of mesenchymal cells into corneal-specific cells, keratocytes. Corneal tissue regeneration is the process by which the cornea repairs and replaces damaged structures. It is a complex process with specific mechanisms. Following corneal injury, epithelial cytokines, chemokines, and growth factors, such as transforming growth factor beta (TGF-β) and platelet-derived growth factor (PDGF), cross the damaged epithelial basement membrane (EBM) and activate quiescent keratocytes in the stroma. These keratocytes transdifferentiate into active, opaque, light-scattering corneal myofibroblasts, which contribute to wound repair by depositing high levels of extracellular matrix components, collagens, and α-actin smooth muscle (α-SMA) stress fibers.Once corneal healing is achieved, it is necessary to remove myofibroblasts from the stroma to restore transparency. However, severe corneal injuries often result in excessive generation and persistence of myofibroblasts and irregular deposition of extracellular matrix components, ultimately compromising corneal transparency.

[0069] It was observed that both the scaffold (1) of the invention (S5N) and the acid scaffold (S5) supported the survival and differentiation of adipose-derived stem cells (ADSCs) into keratocytes over a period of 21 days, during which a progressive change in ADSC morphology was evident, consistent with differentiation into keratocytes. Unlike the acid scaffold (S5), the neutralized scaffold (1) of the invention (S5N) showed moderate degradation by day 7, which did not progress, achieving satisfactory differentiation results by day 21. This phenomenon could be attributed to the fact that keratocytes produce collagenases, enzymes that specifically degrade collagen. Keratocytes tend to preferentially degrade neutral collagen, as it is more abundant and plays a crucial role in maintaining the structural integrity of the corneal extracellular matrix.In contrast, acidic collagen may be less susceptible to degradation due to its unique structural properties and potential modifications that make it more resistant to enzymatic activity. Thus, keratinocytes preferentially degrade neutral collagen to facilitate tissue remodeling and repair.

[0070] Cellular phenotype is a significant factor, particularly in stromal remodeling, where quiescent keratocytes are non-contractile and produce minimal amounts of matrix components. Corneal keratocytes are typically quiescent under normal conditions and are essential for maintaining corneal transparency. Their main specific markers include keratocan and COL I; however, because COL I is a component of the bioink, COL VI was used as an alternative marker. In response to injury, keratocytes are activated by cytokines and growth factors, which can lead to their differentiation into metabolically active myofibroblasts that contribute to tissue repair. Myofibroblasts are highly contractile and produce various matrix proteins and metalloproteinases (MMPs).In their activated state as myofibroblasts, they also produce COL III, a collagen variant that is rapidly deposited in the initial phases of wound healing and is progressively replaced by COL I as repair progresses. Immunofluorescence results in both scaffolds demonstrated that the ADSCs effectively differentiated into keratocytes, as evidenced by the presence of collagen VI and keratocan, confirming that the bioinks did not interfere with keratocyte differentiation or compromise the maintenance of their extracellular matrix. Figure 6 shows, on the one hand, the results of double immunofluorescence (collagen VI and keratocan) captured by confocal microscopy of EU39 ADSCs in passage 1, seeded with KDM and cultured for 21 days in bioinks; and on the other hand, the control group of EU16 donor ADSCs in passage 2, cultured for 20 days with KDM in culture plastic.On the left, DAPI staining highlights cell nuclei; in the center, collagen VI and keratocan staining; on the right, the fused image of both markers. Scale bars represent 20 pm, as indicated in each image. Immunofluorescence results for acidic ink (INK 5) were positive for collagen VI and keratocan, but negative for collagen III, indicating that ADSCs seeded in the bioinks and cultured on KDM differentiated successfully into keratocytes. The negative result for collagen III suggests no differentiation or activation into fibroblasts and myofibroblasts. When the experiment was performed with ADSCs cultured in pH-neutral bioinks (INK 5_NEU) on KDM, the cells also successfully differentiated into keratocytes on the scaffolds.Positive results for collagen VI and keratocan indicated effective differentiation and stability in the matrix secretion characteristics of the keratocytes. Furthermore, a small percentage of cells were positive for collagen III, which, along with the presence of cells negative for collagen VI, suggests activation and differentiation into fibroblasts and myofibroblasts. In this regard, Figure 7 shows, on the one hand, the results of double immunofluorescence (collagen VI and collagen III) captured by confocal microscopy of EU39 ADSCs in passage 9, seeded with KDM and cultured for 21 days in bioinks. On the left, DAPI staining that highlights the cell nuclei; in the center, collagen VI and III; and on the right, the fused staining; and the control group of EU16 ADSCs in passage 2 seeded with KDM and cultured for 20 days in culture plastic. Scale bars represent 20 pm, as indicated in the images.In Figure 8, the cellular stages of the scaffold (1) of the invention (S5N) are graphically represented by their respective percentages, compared to the control. Specifically, the quantification of the double immunofluorescence staining for keratocan / collagen VI and keratocan / collagen III is shown, expressed as percentages. The asterisk indicates statistically significant differences with p < 0.05.

[0071] As shown in Figure 8, the number of undifferentiated ADSCs was generally higher in the scaffold (1) of the invention (S5N); however, this difference was not statistically significant compared to the culture plastic (Figure 8D and G). Similarly, the number of keratocan- and COL VI-positive keratocytes did not show statistically significant differences (Figure 8F), nor did the number of keratocan-positive but COL VI-negative cells (possibly activated keratocytes). In contrast, statistically significant differences were observed in the number of keratocan-negative and COL III-positive cells (fibroblasts and myofibroblasts), with higher counts in the scaffold (1) of the invention (S5N), suggesting a greater tendency for activated keratocyte differentiation in the environment of this scaffold (Figure 8C).In summary, the percentage of undifferentiated ADSCs was approximately 30% in the scaffold (1) of the invention (S5N), as shown in Figure 8D, while differentiation into keratocytes also represented around 30% (Figures 8A and F). The percentage of possibly activated keratocytes was approximately 15% (Figure 8B), and fibroblasts and myofibroblasts were present at a minimum percentage of approximately 3% (Figure 8C). These results demonstrate the potential of the bioink of the invention to support the differentiation of ADSCs into keratocytes.

[0072] Cell Viability

[0073] Finally, the bioink of the invention (INK 5_NEU) was printed with embedded cells. Immortalized human corneal keratocytes (im-hCK) were used, cultured in P60108 fibroblast growth medium supplemented with 1% (v / v) penicillin / streptomycin, 1% (v / v) fibroblast growth supplement, and 10% (v / v) FBS (Innoprot). The viability of the im-hCK cells seeded into each collagen mass was verified using a Live / Dead™ Viability / Cytotoxicity Kit, based on calcein-AM (green, live cells) and ethidium-1 homodimer (red, dead cells). The cell scaffolds were treated according to the manufacturer's instructions, and samples were captured using an inverted fluorescence microscope (Nikon AZ100). Cell viability assays were performed using a concentration of 5 million cells / ml.This test was performed exclusively with the neutralized bioink of the invention (INK 5_NEU), because its neutral pH is more suitable for cell growth.

[0074] Figure 9 presents two key components: a live-dead assay graph and a light transmittance assay. The live-dead assay (cell density of 5 million cells per ml of ink) assesses the survival of the 3D bioprinted scaffolds using im-HK over time, with representative images captured on days 1, 7, and 14. On day 1, predominantly red-stained dead cells are observed, indicating low viability immediately after printing; while on day 7, a constant number of live cells is shown with a slight decrease in red-stained dead cells. By day 14, viability increases to over 80%. Along with the images, a quantitative graph illustrates the percentage of live and dead cells over the 14 days. Embedded cells can remodel the scaffold by applying tension and releasing molecules from the extracellular matrix.In this case, the bioink of the invention (INK 5_NEU) contains COL I and AHA, both components of real tissue, so the im-HK are able to degrade the scaffold components that are part of the stromal ECM, promoting tissue infiltration.

[0075] The light transmittance assay measures the transparency of the neutralized scaffold (1) of the invention (S5N) at the same time points. This shows a decrease in transparency during the first 7 days, likely due to interactions between the cells and the scaffold that affect light transmission. However, from day 14 onward, an improvement in light transmittance is observed, suggesting that the optical clarity of the scaffold recovers as the cells continue to integrate and the scaffold stabilizes. Cells in the stroma also influence the cornea's ability to allow the passage of visible light. Crystalline proteins present in the cytoplasm, such as ALDH1A1 and ALDH3A1, reduce the ability of keratocytes to scatter light. When these cells are activated, as occurred in the previous immunofluorescence assay, the presence of these proteins is reduced, and the cells interfere with light transmission.To improve these results, biochemical signals can be incorporated into the scaffold to stimulate a chemotactic response, thereby enhancing cell migration and growth within the scaffold. Conclusion.

[0076] All the above tests demonstrate that the scaffold (1) of the invention (S5N) is biocompatible with human eye cells, allows cell growth while maintaining its optical properties, making it a suitable solution for corneal tissue regeneration.

[0077] Freeze-drying and sterilization

[0078] Subsequently, the scaffold (1) is freeze-dried (Figure 10) and sterilized with ethylene oxide (EtO). Sterilization with EtO is a recommended option due to its ability to eliminate microorganisms without causing thermal or chemical degradation, as it operates at low temperatures and does not generate ionizing radiation, thus preserving the integrity of the material. Methods such as high-temperature steam or gamma radiation can cause protein denaturation, structural changes, or loss of mechanical and biological properties. For these reasons, ethylene oxide allows the scaffold (1) to maintain its structure, functionality, and biocompatibility.

[0079] The EtO sterilization process begins with packaging the lyophilized scaffold (1) in autoclaving bags, which allow gas penetration and ensure material protection. Next, preconditioning is performed under controlled temperature and humidity to optimize the EtO's effectiveness. In an airtight chamber, the scaffold (1) is exposed to the gas at a temperature of 37–55 °C for 1–6 hours. Subsequently, a prolonged aeration stage is carried out to eliminate toxic EtO residues, ensuring safety. Finally, the process is validated using chemical and biological indicators and sterility tests, verifying that the hydrogel's properties remain unaffected.

[0080] To verify the safety of this sterilization technique and ensure that the scaffold (1) of the invention (S5N) retains its biological properties, direct and indirect biocompatibility tests are performed according to ISO 10993-5. Figure 11A shows that the biocompatibility result is greater than 75% in both tests. This sterilization technique is non-toxic to inks. After rehydration, it maintains its transparency, as shown in Figure 11B, provided it is hydrated with buffered saline solution. When hydrated with calcium chloride, its light transmittance in the visible spectrum decreases. It also maintains adequate thickness, consistency, and appearance. Figure 11C shows the scaffold (1) rehydrated with saline solution, which has a thickness of 320 microns.

Claims

CLAIMS 1. Bio-ink for 3D printing of a scaffold (1) suitable for corneal tissue regeneration, characterized in that it comprises sodium alginate, hyaluronic aldehyde, N,O-carboxymethylated chitosan, type I bovine collagen and sodium hydroxide, in the following concentrations:

2. Bio-ink according to claim 1, wherein the collagen is neutralized by the addition of 20 pl / ml of Sodium Hydroxide (NaOH).

3. Method for preparing the bio-ink of claim 1, comprising: Dissolve each of the components individually, except for collagen, in a phosphate-buffered saline solution, without calcium or magnesium. Combine sodium alginate (ALG) with hyaluronic aldehyde (AHA) and N,O-carboxymethylated chitosan (CMC). Cool the resulting mixture to 4 o C Simultaneously, neutralize the collagen using a 1M NaOH solution at a temperature of 4°C Combine the mixture with the neutralized collagen using a closed syringe system (2) Heat the resulting hydrogel to 37°C for 15 minutes 4. Scaffold (1) suitable for corneal tissue regeneration, characterized by being composed of the bio-ink of claim 1.

5. Scaffold (1) according to claim 4, the composition of which further includes live cells of corneal origin.

6. Method of printing the anadamium (1) of claims 4 and 5, using a 3D extrusion bioprinter, comprising: Load the bio-ink into a 30cc UV-protected cartridge, equipped with a piston and a 25G blunt needle. Configure the bioprinter with the following printing parameters: 10 kPa pressure, at a temperature of 30°C and a speed of 1 mm / s. For the characterization of the scaffolding, use a cylinder 10 mm in diameter and 0.5 mm in height.

7. Method for sterilizing the scaffold (1) of claims 4 and 5, comprising: Pack the freeze-dried scaffold (1) into autoclaving bags. Subject the scaffold (1) to preconditioning at controlled temperature and humidity. In an airtight chamber, expose the scaffold (1) to EtO gas at a temperature of 37-55 °C for 1-6 hours. Subject the scaffold (1) to prolonged aeration to remove toxic residues. Rehydrate the scaffold (1), using a buffered saline solution

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