A lenticule, method of preparation and implementations thereof

Abstract

The present disclosure provides a three-dimensional hydrogel prepared using a bioink composition comprising methacrylated hyaluronic acid, methacrylated gelatin, a photoinitiator and a photoabsorber; and a method for preparing the three- dimensional hydrogel or lenticule. The present disclosure further provides a method of treating corneal tissue repair in a subject in need thereof using the three- dimensional hydrogel or lenticule.

Description

A LENTICULE, METHOD OF PREPARATION AND IMPLEMENTATIONS THEREOFFIELD OF INVENTION

[0001] The present disclosure broadly relates to the field of hydrogels for biomedical applications. The present disclosure particularly relates to a three-dimensional hydrogel or lenticule prepared using a bioink composition.BACKGROUND OF THE INVENTION

[0002] According to the World Health Organization (WHO), approximately 1.9 million individuals suffer from corneal blindness caused by corneal opacification ( S. C. Tidke and P. Tidake, “A Review of Corneal Blindness: Causes and Management, ” Cureus, Oct. 2022, doi: 10.7759 / cureus.30097). This condition constitutes approximately 5% of all blindness cases worldwide. Corneal blindness results from the progressive development of several conditions, including infectious ones such as herpes simplex keratitis, bacterial keratitis, and fungal keratitis. Additionally, non-infectious uveitis, glaucoma, trachoma, keratoconus, dry eye disease, and corneal abrasions arising from trauma or object entering the eyes also contribute to the prevalence of corneal blindness (J. D. Steinmetz et al., “Causes of blindness and vision impairment in 2020 and trends over 30 years, and prevalence of avoidable blindness in relation to VISION 2020: the Right to Sight: an analysis for the Global Burden of Disease Study, ” Lancet Glob Health, vol. 9, no. 2, pp. el44-el60, Feb. 2021, doi: 10.1016 / S2214-109X(20)30489- 7 ). These conditions can have serious consequences if they are not detected and treated quickly. Hence the discovery of effective treatment holds significant importance.

[0003] Keratoplasty is currently the gold standard of care for corneal transplantation. Penetrating keratoplasty (PKP), deep anterior lamellar keratoplasty (DALK), and endothelial keratoplasty (EK) are the three major techniques that are used to target specific layers of the cornea and patient requirements (G. E. Boynton and M. A. Woodward, “Evolving Techniques in Corneal Transplantation, ” Curr Surg Rep, vol.3, no. 2, p. 2, Feb. 2015, doi: 10.1007 / s40137 -014-0079-5). PKP involves the entire damaged or diseased cornea being replaced with a healthy donor cornea (D. T. H. Tan et al., “Penetrating Keratoplasty in Asian Eyes, ” Ophthalmology, vol. 115, no. 6, pp. 975-982.el, Jun. 2008, doi: 10.10167j.ophtha.2007.08.049; B. Gurnani and K. Kaur, Penetrating Keratoplasty. 2023). PKP is usually adopted in cases of advanced keratoconus, corneal scarring, and corneal dystrophies. Transplantation often comes with the risk of rejection, and to mitigate these risks the patients require long-term immunosuppressive medications, which may have their own set of side effects and complications. PKP also leads to astigmatism which ultimately affects the final visual outcomes. DALK is another procedure where the anterior layers of the cornea are replaced while the endothelial layers are kept intact. DALK is preferable in the early stages of keratoconus and corneal injuries that do not involve the endothelial layers. DALK demands precise surgical skills as separating the layers of the cornea could be challenging. Descemet's membrane (DM) should not be perforated during DALK as any injury to the DM would require PKP (A. Anshu, M. O. Price, and F. IT. Price, “Risk of Corneal Transplant Rejection Significantly Reduced with Descemet’s Membrane Endothelial Keratoplasty, ” Ophthalmology, vol. 119, no. 3, pp. 536 540, Mar. 2012, doi: 10.10167j.ophtha.2011.09.019). DALK is also associated with a postoperative haze that again impairs the visual outcome. Endothelial keratoplasty involves selective replacement of the damaged endothelial layer of the cornea with a donor endothelial layer. Descemet's Stripping Automated Endothelial Keratoplasty (DSAEK) and Descemet's Membrane Endothelial Keratoplasty (DMEK) are two procedures that come under the bracket of endothelial keratoplasty (EK) ( B. D. Allan, M. A. Terry, F. W. Price, M. O. Price, N. B. Griffin, and M. Claesson, “Corneal Transplant Rejection Rate and Severity After Endothelial Keratoplasty, ” Cornea, vol. 26, no. 9, pp. 1039-1042, Oct. 2007, doi: 10.1097 / ICO.0b013e31812f66e5; F. IT. Price and M. O. Price, “Descemet ’s Stripping With Endothelial Keratoplasty in 50 Eyes: A Refractive Neutral Corneal Transplant, ” Journal of Refractive Surgery, vol. 21, no. 4, pp. 339-345, Jul. 2005, doi: 10.3928 / 1081-597X-20050701-07). EK has aslight edge over PKP and DALK as the visual outcomes are improved due to reduced astigmatism. In the early postoperative period, EK may experience dislocation and may require re-transplantation. Apart from the inherent limitations of each technique, researchers have explored xenotransplantation where corneas from genetically modified pigs have been investigated. However, they possess significant immunological challenges and a risk of disease transmission. Corneal transplantation still remains the standard procedure for several corneal diseases and conditions. Significant challenges such as donor shortage, graft rejection, and postoperative complications continue to drive ongoing research and innovation in this field. Addressing these limitations is crucial in order to improve outcomes and foster innovations in corneal regeneration.

[0004] Cornea is the transparent, outermost dome-shaped layer that allows light to enter the eye and focus on the retina. It is responsible for about two-thirds of the eye's total optical power and contributes significantly to image clarity and visual acuity. When designing a cornea substitute or artificial cornea (keratoprosthesis), it is essential to mimic the native properties of the cornea to ensure successful integration and functional performance (M. Rafat et al., “Bioengineered corneal tissue for minimally invasive vision restoration in advanced keratoconus in two clinical cohorts, ” Nat Biotechnol, vol. 41, no. 1, pp. 70—81, Jan. 2023, doi: 10.1038 / s41587-022-01408-w). However, it is worth noting that despite significant advancements in this field, the development of a fully functional and widely accessible artificial cornea remains a complex and ongoing challenge.SUMMARY OF INVENTION

[0005] In an aspect of the present disclosure, there is provided a three-dimensional hydrogel comprising a plurality of layers forming a three-dimensional structure, wherein each layer is a polymeric scaffold comprising cross-linked methacrylated hyaluronic acid and methacrylated gelatin.

[0006] In another aspect of the present disclosure, there is provided a method for preparing the three-dimensional hydrogel as disclosed herein, wherein the process comprising: preparing a bioink composition comprising methacrylated hyaluronic acid, methacrylated gelatin, photoinitiator, and photoabsorber in an aqueous carrier; and fabricating the three-dimensional hydrogel, layer-by-layer, wherein each layer is separately exposed to photoirradiation for cross-linking of bioink.

[0007] In yet another aspect of the present disclosure, there is provided a method of treating a corneal tissue repair in a subject in need thereof, comprising implanting the three-dimensional hydrogel as disclosed herein in the corneal tissue.

[0008] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

[0009] The following drawings form a part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

[0010] Figure 1 depicts the schematic presentation of bioink composition preparation and digital light processing (DLP) bioprinting of HA-MA / Gel-MA based corneal lenticule, in accordance with the embodiments herein.

[0011] Figure 2 depicts the CAD model of the corneal lenticules (a-b), photograph of printed lenticule (c) and (d) stereomicroscope images of the bioprinted lenticule, in accordance with the embodiments herein.

[0012] Figure 3 depicts the photographic images of corneal lenticules printed at 2 2 different projection power outputs a) 27.86 mW / cm , and b) 31.06 mW / cm , in accordance with the embodiments herein.

[0013] Figure 4 depicts mechanical properties of the bioprinted lenticules, (a-b) adhesion Strength, (c-d) puncture resistance, and (e-f) drag strength, in accordance with the embodiments herein.

[0014] Figure 5 depicts the viability of human corneal stromal cells evaluated on 2D surfaces (a & b), within 3D bioprinted lenticules (c-f) and the z-axis projection (g-j). Scale bar 200 pm, in accordance with the embodiments herein.

[0015] Figure 6 depicts the results of cells subjected to phenotype evaluation, hCSCs cultured on a coverslip (a-b), and those encapsulated within 3D bioprinted lenticules (c-f). Scale bar 200 pm., in accordance with the embodiments herein.

[0016] Figure 7 depicts the phase contrast images of cells on 2D coverslip (a), and 3D bioprinted lenticule (c) surface. Scale bar 200 pm. Immunofluorescence staining was done for ZO-1 (green), Rhodamine-phalloidin (red) and DAPI (blue) on 2D coverslip (b), and 3D bioprinted lenticule (d, e). Scale bar 100 pm., in accordance with the embodiments herein.DETAILED DESCRIPTION OF THE INVENTION

[0017] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps, features, compositions, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features.Definitions

[0018] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are delineated here. Thesedefinitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.

[0019] The articles “a”, “an” and “the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0020] The terms “comprise” and “comprising” are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as “consists of only”.

[0021] Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.

[0022] The term “including” is used to mean “including but not limited to”. “Including” and “including but not limited to” are used interchangeably.

[0023] The terms “w / w”, as used herein, refers to percentage by weight, relative to the weight of the total composition, unless otherwise specified.

[0024] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference.

[0025] Timely identification and prompt intervention play a crucial role in minimizing harm and enhancing individuals' quality of life with corneal blindness. Moreover, new and emerging treatments, such as those utilizing regenerative medicine, offer more hope for the future. Rapid advancement in tissue fabrication techniques has laid the pathway to overcome the drawbacks of traditional tissue engineered scaffolds such as inability to replicate well-defined complex tissue structures (G. Gao and X. Cui,“Three-dimensional bioprinting in tissue engineering and regenerative medicine, ” Biotechnology Letters, vol. 38, no. 2. Springer Netherlands, pp. 203—211, Feb. 01, 2016. doi: 10.1007 / sl0529-015-1975-l.) On that note, three-dimensional (3D) bioprinting has altered the paradigm of tissue engineering by emerging as a promising candidate for facile biofabrication of patient-specific delicate tissue structures (B. Tan, S. Gan, X. Wang, W. Liu, and X. Li, “Applications of 3D bioprinting in tissue engineering: advantages, deficiencies, improvements, and future perspectives, ” Journal of Materials Chemistry B, vol. 9, no. 27. Royal Society of Chemistry, pp. 5385— 5413, Jul. 21, 2021. doi: 10.1039 / dltb00172h ). The diverse portfolio of 3D bioprinting techniques utilized till today for fabricating artificial cornea comprises of extrusionbased bioprinting, stereolithography (SLA) or DLP -based bioprinting, Inkjet bioprinting and Laser-assisted bioprinting (M. Ben Thomas et al., “Print me a cornea - Are we there yet?, ” Bioprinting, vol. 28. Elsevier B. V., Dec. 01, 2022. doi: 10.1016 / j.bprint.2022.e00227). Despite these varied methods, it is crucial to have a delicate balance of the material chosen for bioprinting and precise printability of the structural dimensions, which ultimately determines the functionality of the 3D bioprinted cornea. The present disclosure provides a DLP based fabricating of hydrogel structure, a corneal lenticule, with intricate geometries and high bioprinting resolution that mimics the cornea.Three-dimensional hydrogel

[0026] Embodiments herein include a three-dimensional hydrogel. In an embodiment of the present disclosure, there is provided a three-dimensional hydrogel comprising a plurality of layers forming a three-dimensional structure, wherein each layer is a polymeric scaffold comprising cross-linked methacrylated hyaluronic acid and methacrylated gelatin.

[0027] In an embodiment of the disclosure, the polymeric scaffold is obtained using a bio-ink composition comprising, in an aqueous carrier: a) methacrylated hyaluronic acid; b) methacrylated gelatin; c) a photoinitiator; and d) a photoabsorber.

[0028] In an embodiment of the present disclosure, the bio-ink composition further comprises a plurality of cells, drugs, extracellular vesicles, nanovesicles, or mixtures thereof. In an embodiment of the present disclosure, the plurality of cells may be MSCs (Mesenchymal stem cells). In an embodiment of the present disclosure, the MSCs may be human MSCs. In an embodiment of the present disclosure, the human MSCs are selected from human corneal stromal cells (hCSCs), keratocytes, corneal epithelial cells, or corneal endothelial cells.

[0029] In an embodiment of the present disclosure, the methacrylated hyaluronic acid is in a weight percentage range of 1.81 to 6.35% (20-70 mg / ml); and methacrylated gelatin is in a weight percentage range of 1.81 to 10.88% (20-120 mg / ml), in respect of the bioink composition. In another embodiment of the present disclosure, the methacrylated hyaluronic acid is in a weight percentage range of 2.71 to 6.33% (30-70 mg / ml), 2.71 to 5.42% (30-60 mg / ml), 3.62 to 6.33% (40-70 mg / ml), 3.62 to 5.42% (40-60 mg / ml), or 4.07 to 5.42% (45-60 mg / ml), in respect of the bioink composition; and methacrylated gelatin is in a weight percentage range of 2.71 to 9.95% (30-110 mg / ml), 4.52 to 9.04% (50-100 mg / ml), 4.52 to 8.14% (50-90 mg / ml), 4.52 to 7.24% (50-80 mg / ml), 4.52 to 6.33% (50-70 mg / ml), or 4.52 to 5.42% (50-60 mg / ml) in respect of the bioink composition.

[0030] In a first exemplary embodiment, the methacrylated hyaluronic acid is in a weight percentage range of 2.71 to 6.33% (30-70 mg / ml), and methacrylated gelatin is in a weight percentage range of 4.07 to 5.42% (45-60 mg / ml), in respect of the bioink composition. In a second exemplary embodiment, the methacrylated hyaluronic acid is in a weight percentage of 4.52 to 4.97% (55 mg / ml), and methacrylated gelatin is in a weight percentage of 4.07 (45 mg / ml), in respect of the bioink composition. In a third exemplary embodiment, the methacrylated hyaluronic acid is in a weight percentage of 5.42 % (60 mg / ml), and methacrylated gelatin is in a weight percentage of 5.42% (60 mg / ml), in respect of the bioink composition. In a fourth exemplary embodiment, the methacrylated hyaluronic acid is in a weight percentage of 4.52 % (50 mg / ml), and methacrylated gelatin is in a weight percentage of 4.52 (50 mg / ml), in respect of thebioink composition. In a fifth exemplary embodiment, the methacrylated hyaluronic acid is in a weight percentage of 2.71 % (30 mg / ml), and methacrylated gelatin is in a weight percentage of 6.33 (70 mg / ml), in respect of the bioink composition.

[0031] In an embodiment of the present disclosure, the ratio of methacrylated hyaluronic acid to the methacrylated gelatin is in a range of 1:0.2 to 1:6, 1:0.5 to 1:5, 1:0.7 to 1:4, 1:0.8 to 1:3 or 1:0.8 to 1:2.5. In preferred embodiments of the present disclosure, the ratio of methacrylated hyaluronic acid to the methacrylated gelatin is 1:1, 1:2.3 or 1:0.8.

[0032] In an embodiment of the present disclosure, the degree of substitution of methacrylate groups in the methacrylated hyaluronic acid is in a range of 20 to 75%; and the degree of substitution of methacrylate groups in the methacrylated gelatin is in a range of 40 to 99%. In another embodiment of the present disclosure, the degree of substitution of methacrylate groups in the methacrylated hyaluronic acid is in a range of 20 to 75%, 20 to 70%, 30 to 70%, 40 to 70%, 40 to 60%, 40 to 50%, or 50 to 60%; and the degree of substitution of methacrylate groups in the methacrylated gelatin is in a range of 40 to 90%, 40 to 85%, 40 to 75%, 40 to 70%, 40 to 60%, 50 to 60%, 50 to 90%, 60 to 90%, or 60 to 80%.

[0033] In an embodiment of the present disclosure, the molecular weight of hyaluronic acid in the methacrylated hyaluronic acid is in a range of 5 to 60 kDa. In another embodiment of the present disclosure, the molecular weight of hyaluronic acid in the methacrylated hyaluronic acid is in a range of 10-50 kDa, 20 to 50 kDa, 30 to 50 kDa, 20 to 40 kDa, or 30 to 40 kDa.

[0034] In an embodiment of the present disclosure, the molecular weight of gelatin in the methacrylated gelatin is in a range of 50 to 1000 kDa. In another embodiment of the present disclosure, the molecular weight of gelatin in the methacrylated gelatin is in a range of 50 to 900 kDa, 50 to 800 kDa, 50 to 500 kDa, 50 to 400 kDa, 50 to 200 kDa, 50 to 150 kDa, 50 to 120 kDa, 50 to 100 kDa, 60 to 100 kDa, 70 to 100 kDa, or 70 to 90 kDa.

[0035] In an exemplary embodiment of the present disclosure, the molecular weight of hyaluronic acid in the methacrylated hyaluronic acid is 33 kDa, and the molecular weight of gelatin in the methacrylated gelatin is 90 kDa.Photoinitiator

[0036] The bioink composition, according to embodiments herein, includes a photoinitiator.

[0037] The term “photoinitiator”, as used in the present disclosure, refers to a substance that creates reactive species (free radicals, cations, or anions) when exposed to radiation (UV or visible). They initiate the polymerization process by adding themselves to the monomer or oligomer. In various embodiments herein, it refers to the molecules which facilitates the formation of cross-linked polymeric scaffolds of biopolymers as part of the composition.

[0038] Various photoinitiators are known and may be used in various embodiments herein. In an embodiment of the present disclosure, the composition disclosed herein includes a photoinitiator selected from sodium persulphate (SPS), ruthenium, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), riboflavin, 2-Hydroxy-4'-(2- hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959), or mixtures thereof. In another embodiment, the photoinitiator is a mixture or combination of sodium persulphate (SPS) and ruthenium particularly, chloro(pentamethylcyclopentadienyl)bis(triphenylphosphine)ruthenium(II).

[0039] In an embodiment of the present disclosure, the photoinitiator is in a weight percentage range of 0.005 to 0.7% (1-30 mM), in respect of the bioink composition. In another embodiment, the photoinitiator is in a weight percentage range of 0.007 to 0.6%, 0.009 to 0.55%, 0.07 to 0.5%, 0.07 to 0.32%, 0.09 to 0.45%, 0.1 to 0.4%, 0.15 to 0.35%, 0.18 to 0.33%, or 0.2 to 0.32%.

[0040] In a preferred embodiment of the present disclosure, the photoinitiator is a combination of sodium persulphate (SPS) and chloro(pentamethylcyclopentadienyl)bis(triphenylphosphine)ruthenium(II) . In another preferred embodiment, sodium persulphate (SPS) is in a weight percentage range of0.15 to 0.3%, 0.17 to 0.25%, or 0.18 to 0.24%, in respect of the bioink composition; and chloro(pentamethylcyclopentadienyl)bis(triphenylphosphine)ruthenium(II) is in a weight percentage range of 0.05 to 0.09%, 0.06 to 0.08% or 0.065 to 0.075%, in respect of the bioink composition. In an exemplary embodiment, sodium persulphate (SPS) is in a weight percentage of 0.238% (lOmM), in respect of the bioink composition, and chloro(pentamethylcyclopentadienyl)bis(triphenylphosphine)ruthenium(II) is in a weight percentage of 0.075% (ImM), in respect of the bioink composition.Photoabsorber

[0041] The composition, according to embodiments herein, includes photoabsorber.

[0042] The term “photoabsorber”, as used in the present disclosure, refers to a substance that is used to control the penetration depth of incident light in polymerizable material. In various embodiments herein it refers to the molecules, which prevents the excessive cross-linking of polymerizable material, for e.g.: biopolymers.

[0043] Various photoabsorbers are known and may be used in various embodiments herein. In an embodiment of the present disclosure, the composition disclosed herein includes a photoabsorber selected from a photoabsorber dye selected from tartrazine, fast green FCF (FD&C Green No. 3 dye), new coccine, methylene blue, or mixtures thereof.

[0044] In an embodiment of the present disclosure, the photoabsorber is in a weight percentage range of 0.007 to 0.04%, 0.01 to 0.04%, 0.015 to 0.03%, or 0.02 to 0.03, in respect of the bioink composition. In a preferred embodiment, the photoabsorber is tartrazine present in a weight percentage range of 0.007 to 0.04%, 0.01 to 0.04%, 0.015 to 0.03%, or 0.02 to 0.03%, in respect of the bioink composition.Aqueous carrier

[0045] In an embodiment of the present disclosure, the composition comprises an aqueous carrier. In another embodiment, the aqueous carrier is selected from phosphate buffered saline, saline, water (optionally distilled water), or mixtures thereof.

[0046] In an embodiment of the present disclosure, the aqueous carrier is in a weight percentage range of 70 to 95%, in respect of the composition.

[0047] Embodiments herein include a three-dimensional hydrogel comprising a plurality of layers. A person of skill in the art will understand that the parameter such as height of the layer will be dependent on the type of the instrument or equipment used, since different instruments have different capabilities and limitations.

[0048] In an embodiment of the present disclosure, each of said layer is characterized by a height in the range of 10 to 200 pm; and the number of layers is in a range of 20 to 400. In another embodiment of the present disclosure, each of said layer is characterized by a height in the range of 50 to 150 pm; and the number of layers is in a range of 25 to 200. In yet another embodiment of the present disclosure, each of said layer is characterized by a height in the range of 80 to 120 pm; and the number of layers is in a range of 30 to 80. In one or more embodiments of the present disclosure, each of said layer is characterized by a height in the range of 80 to 110 pm; and the number of layers is in a range of 30 to 50. In a preferred embodiment, each of said layer is characterized by a height in the range of 90 to 110 pm; and the number of layers is in a range of 30 to 40.

[0049] In an embodiment of the present disclosure, the three-dimensional hydrogel encapsulates a plurality of cells drugs, extracellular vesicles, nanovesicles, or mixtures thereof. In an embodiment of the present disclosure, the plurality of cells may be MSCs Mesenchymal stem cells (MSCs). In an embodiment of the present disclosure, the MSCs may be human MSCs. In an embodiment of the present disclosure, the human MSCs are selected from human corneal stromal cells (hCSCs), keratocytes, corneal epithelial cells, or corneal endothelial cells.

[0050] Characteristics of the hydrogel

[0051] In an embodiment of the present disclosure, the hydrogel is characterized by a transmittance in a range of 80% to 94%. In another embodiment of the present disclosure, the hydrogel is characterized by a transmittance in a range of 80% to 92%, 80% to 90%, 80% to 88% or 80% to 85%.

[0052] In an embodiment of the present disclosure, the hydrogel is characterized by a mass swelling ratio in a range of 1 to 45%, 1 to 40%, 1 to 35%, 1 to 20%, 2 to 30%, 2 to 28%, 2 to 25%, or 2 to 20%.

[0053] In an embodiment of the present disclosure, the hydrogel is characterized by a compressive modulus in a range of 400 kPa to 600 kPa, 450 kPa to 600 kPa, 480 kPa to 600 kPa, 500 kPa to 600 kPa, 500 kPa to 580 kPa, 500 kPa to 560 kPa, or 500 kPa to 550 kPa.

[0054] In an embodiment of the present disclosure, the hydrogel is characterized by an adhesion strength of 30 kPa to 80 kPa, 30 kPa to 70 kPa, 30 kPa to 60 kPa, or 40 kPa to 60 kPa.

[0055] In an embodiment of the present disclosure, wherein the three-dimensional hydrogel, under in vitro conditions, has about 15% degradation within 28 to 30 days.

[0056] In an embodiment of the present disclosure, there is provided a three- dimensional hydrogel in the form of a lenticule, particularly, a corneal lenticule.Method of preparing the three-dimensional hydrogel

[0057] Embodiments herein further include a method of preparing the three- dimensional hydrogel disclosed in various embodiments herein.

[0058] In an embodiment of the present disclosure, the method for preparing the hydrogel comprises preparing a bioink composition comprising methacrylated hyaluronic acid, methacrylated gelatin, photoinitiator, and photoabsorber in an aqueous carrier; and fabricating the three-dimensional hydrogel, layer-by-layer, wherein each layer is separately exposed to photoirradiation for cross-linking of bioink, particularly cross-linking of methacrylated hyaluronic acid and methacrylated gelatin. In another embodiment, the layer-by-layer fabrication is performed by a digital light processing printer.

[0059] Digital light processing printer uses digital light processing technology for fabricating a 3D structure. In the present disclosure, a digital light processing printer has been used to fabricate the three-dimensional hydrogel as described herein. In a Digital Light Processing (DLP) bioprinter, layers are arranged sequentially to fabricatea corneal lenticule by utilizing a photo-crosslinkable bioink. To produce a given layer, a unit of the bioink is applied on a surface, which may be a previously formed layer, and is photoirradiated to induce cross-linking so that the bioink is converted into a cross-linked scaffold, and these steps are repeated for each layer until a desired number of layers are formed. Thus, each new flat cylindrical layer merges with the previous layer, creating a seamless lenticular structure, termed as a lenticule. The layers can be designed to mimic the cornea’s natural curvature and complex shape, incorporating varying thicknesses and cell densities for tissue functionality. The layer-by-layer fabrication approach allows the creation of intricate and highly detailed structures, including gradients in cell distribution, which is vital for mimicking the natural structure and function of tissues like the cornea.

[0060] In an embodiment of the present disclosure, photoirradiation comprises subjecting or exposing each layer to photoirradiation. This exposure in the present disclosure is termed as “exposure time per layer”. The exposure time per layer in a 3D digital light processing (DLP) bioprinter refers to the duration that light is applied to each layer of the build material (bioink) during printing. This exposure time plays a key role in determining the quality and accuracy of the printed structure. If the exposure time is too long, it can cause over-curing, leading to blurring or excessive hardening of material. Conversely, an exposure time that is too short may result in incomplete curing, leading to weak or poorly defined layers.

[0061] In an embodiment of the present disclosure, the photoirradiation is carried out using light of intensity in the range of 10 to 35 mW / cm2, 10 to 35 mW / cm2, 15 to 35 mW / cm2, 20 to 35 mW / cm2, 25 to 35 mW / cm2, or 30 to 35 mW / cm2. In another embodiment of the present disclosure, the photoirradiation is carried out at a wavelength in the range of 400 to 700nm, 400 to 600nm, 400 to 500nm, 400 to 450nm, 400 to 430nm, 400 to 415nm, preferably 405nm.

[0062] In an embodiment of the present disclosure, the photoirradiation is carried out for a time period in the range of 0.2 to 30 seconds, 0.5 to 20 seconds, 0.8 to 20 seconds, 1 to 20 seconds, 2 to 15 seconds, 2 to 13 seconds, 2 to 11 seconds, or 2 to 10 seconds.

[0063] In an embodiment of the present disclosure, photoirradiation further comprises exposing the first layer of the print to light, typically the base or bottom layer. This exposure is termed as “base exposure time” or “base exposure factor” in the present disclosure, which in a 3D Digital Light Processing (DLP) bioprinter is the initial duration during which the first layer or the bottom most layer is exposed to light. This exposure time is usually longer than those for the following layers (exposure time per layer) to ensure strong adhesion of the first layer to the build platform and to fully cure it before printing continues with the subsequent layers. In an embodiment of the present disclosure, the base exposure factor is selected from 2X, 3X, 4X and 5X, where in X is the exposure time per layer selected from 0.2 to 30 seconds, 0.5 to 20 seconds, 0.8 to 20 seconds, 1 to 20 seconds, 2 to 15 seconds, 2 to 13 seconds, 2 to 11 seconds, or 2 to 10 seconds, preferably, the exposure time per layer is 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds or 10 seconds.

[0064] In an embodiment of the present disclosure, the method comprises fabricating the three-dimensional hydrogel at power projection output or level in the range of 40% to 80%, 50% to 70%, or 60% to 70%. The projection power level in a 3D digital light processing (DLP) bioprinter refers to the intensity of light projected onto the build surface during printing. This power level is usually controlled by adjusting the brightness or power of the light source (typically a digital projector), affecting the curing speed, precision, and resolution of the printed structure.

[0065] In an embodiment of the present disclosure, the method comprises fabricating the three-dimensional hydrogel at a print bed temperature in the range of 30 to 40 °C, 32 to 38 °C, or 33 to 36 °C. The print bed temperature in a 3D Digital Light Processing (DLP) bioprinter refers to the temperature of the build platform where the material is deposited and cured during printing. This temperature is crucial for ensuring proper adhesion of the printed layers to the surface and also affects the material's behavior during the curing process.

[0066] In an embodiment of the present disclosure, the method comprises, once the desired number of layers are formed, an optional step of removing excess non-cross-linked bioink, and post-curing the three-dimensional hydrogel in the presence of light. Optionally, the light is a blue light between 380 and 450 nm wavelength, and postcuring is carried out for a period in the range of 5 minutes to 20 minutes. In another embodiment, the light is a blue light between 400 and 430 nm wavelength, and postcuring is carried out for a period in the range of 5 minutes to 15 minutes. In yet another embodiment, the light is a blue light between 390 and 410 nm wavelength, and postcuring is carried out for a period in the range of 7 minutes to 12 minutes. In a preferred embodiment, the light is a blue light of 405 nm wavelength (20 mW / cm2), and postcuring is carried out for a period in the range of 8 minutes to 10 minutes.

[0067] In an embodiment of the present disclosure, the method comprises providing a plurality of cells, drugs, extracellular vesicles, nanovesicles or mixtures thereof, to the bioink composition prior to fabricating the three-dimensional hydrogel.

[0068] In an embodiment of the present disclosure, the method for preparing the hydrogel comprises preparing the bioink composition; and fabricating the three- dimensional hydrogel, layer-by-layer, using a digital light processing printer under photoirradiation using light of intensity in the range of 10 to 35 mW / cm2, for a period of time in the range of 0.2 to 30 seconds, and optionally post-curing the three- dimensional hydrogel in the presence of light of 405 nm wavelength (20 mW / cm2) for a period in the range of 5 minutes to 20 minutes. In another embodiment of the present disclosure, the method for preparing the hydrogel comprises preparing the bioink composition; and fabricating the three-dimensional hydrogel, layer-by-layer, using a digital light processing printer under photoirradiation using light of intensity in the range of 10 to 35 mW / cm2, for a period of time in the range of 2 to 4 seconds, and optionally post-curing the three-dimensional hydrogel in the presence of light of 405 nm wavelength (20 mW / cm2) for a period in the range of 8 minutes to 12 minutes.

[0069] In an embodiment of the present disclosure, there is provided a method treating a corneal tissue repair in a subject in need thereof, comprising implanting the three- dimensional hydrogel as disclosed herein in the corneal tissue.

[0070] Although the subject matter has been described with reference to specific embodiments, this description is not meant to be construed in a limiting sense. Various modifications of the disclosed embodiments, as well as alternate embodiments of the subject matter, will become apparent to persons skilled in the art upon reference to the description of the subject matter. It is therefore contemplated that such modifications can be made without departing from the spirit or scope of the present subject matter as defined.EXAMPLES

[0071] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.EXAMPLE 1Preparation and physical characterization of the three-dimensional hydrogels (lenticule)

[0072] Bio-ink composition preparation:

[0073] Working Example: Methacrylated hyaluronic acid (HA-MA, 33kDa, 50% DoS, Blafar) and methacrylated gelatin (Gel-MA, 90kDa, 60% DoS, Rousselot) were used for bio-ink composition preparation (Figure 1). Different working compositions were prepared with varying parameters as illustrated in Table 1 below. Generally, HA-MA and Gel-MA were pre-mixed and dissolved in a predetermined volume of IX PBS. Themixture was then incubated at 40°C on a dry bath under dark condition with intermittent vortexing. Once the polymer mixture was completely dissolved in IX PBS, photoinitiators) and photo absorber was added. The photoinitiators were prepared in PBS. Once all components were completely dissolved in IX PBS, the bio-ink composition was loaded on the vat-plate (PDMS coated plates) in the LumenX bioprinter for printing the lenticules.Table 1. Working Compositions

[0074] Non-Working Example: Several non-working compositions were also prepared with varying parameter such that they fall beyond the desired range (Table 2). The general method used for preparing the composition was same as described hereinabove for the working examples. Table 2. Non-working compositions

[0075] Digital model generation: Prior to printing the lenticules, a digital corneal model was generated based on the dimensions of human cornea (M. Dubbelman, V. A. D. P. Steam, and G. L. Van derHeijde, “The shape of the anterior and posterior surface of the aging human cornea, ” Vision Res, vol. 46, no. 6-7, pp. 993-1001, Mar. 2006, doi: 10.1016 / j.visres.2005.09.021). Based on this, the horizontal diameters, and the radius of curvature of the model was set as 13.99 mm and 3.98 mm respectively while its thickness was 200 pm. The corneal model was created based on Computed Tomography images of human cornea using AUTOCAD® version. The structure was exported as an STL file and was fed into the 3D bioprinter (LumenX, CELLINK / DLP printer) where it was auto sliced by the software into 37 layers with a resolution of 100 pm height per layer.

[0076] DLP 3D printing system configuration and printing process: The pre-designed STL files for corneal lenticules were printed using the bioprinter. The DLP bioprinter (Lumen X, CELLINK) used a layer-by-layer approach for printing with a pixel resolution (XY) of 50 pm and Z-Precision resolution of 5 pm. The print bed temperature was set at 35°C. The light intensity and time were set at indicated in Table 1 and 2 above. Once the printing process was complete, the formed structure wasobtained from the build platform, washed with IX PBS for removing excess non-cross- linked bioink and optionally post cured (blue light, 20 mW / cm2) for 10 minutes.

[0077] Cylindrical hydrogels (diameter: 3 mm, height: 1 mm), for the purpose of studying the characteristics of the hygrogel, were printed using DLP bioprinter for carrying out various mechanical characterizations like compressive modulus measurements, swelling degree measurements and puncture resistance analysis. To print cylindrical hydrogels, power projection output was set as indicated in Table 1 and 2, while the print bed temperature was fixed at 35°C. The base exposure time was 6s while it was 2s for the remaining layers.Compressive Modulus Measurements

[0078] Cylindrical hydrogels were fabricated with specific dimensions (3 mm diameter & 1 mm height) using the LumenX bioprinter. These hydrogels were then subjected to compressive modulus testing using a BiSS mechanical tester with parallel plate fixtures. Before the testing, the height and diameter of the hydrogels were measured. During the testing, the hydrogels were compressed at a controlled rate of 1 mm / min, up to a maximum compression of 50%, using a 10N load cell. The deformation (strain) and applied force (load) were continuously monitored and recorded. The compressive modulus was determined by analyzing the stress-strain curve, focusing on a linear region between 0.1 to 0.2 mm / min strain.Swelling Degree Measurements

[0079] Cylindrical hydrogel discs with specific dimensions (3 mm diameter & 1 mm height) were printed using LumenX bioprinter and then incubated in IX PBS to assess their swelling behavior over time. The weights were recorded at various time intervals. The swelling percentage by weight was calculated using the formula: [(Wt - Wi) / Wi] x 100, where Wt represents the weight recorded at different time points, and Wi is the initial weight of the hydrogel discs.Adhesion strength analysis:

[0080] The adhesion strength of the hydrogels was measured using a modified standard test method for adhesion strength (ASTM F2458-05) (N. Annabi et al., “Engineeringa highly elastic human protein-based sealant for surgical applications, ” Sci Transl Med, vol. 9, no. 410, p. eaai7466, 2017). The study was carried out using porcine skin. The skin segments were thoroughly washed with water and PBS before conducting the experiment. After removing excess moisture from the skin surface, the skin tissue was cut into 2.5 cm by 1.5 cm dimensions, and each piece was affixed to two glass slides with glue. The two glass slides were positioned with a 10 mm gap in between. Once the skin was properly attached to the glass slides, an incision was made with a straightedge razor along the gap to simulate a wound site. Using a custom mould, 75 pl of bioink was applied over the wound site followed by white light- mediated cross-linking step (intensity: 31.0 mW / cm2). After photo cross-linking, the arrangement was immersed in IX PBS for optimum swelling. The samples were then transferred from IX PBS to the BiSS uniaxial mechanical tester, and the adhesive strength of the hydrogels was measured until the point of breakage or cohesive failure of the gels. The applied strain rate was 1 mm / min (n > 3) to determine the adhesive strength, and the data was reported in terms of maximum adhesive strength before failure.Puncture resistance analysis:

[0081] To conduct the puncture test, a 10-0-3 / 8G surgical needle (0.052 mm2) was clamped to wedge action grips with a 44N load cell of the BiSS uniaxial mechanical testing instrument. These wedge action grips, along with the needle was vertically attached above the 3D bio-printed cylinders with dimensions of 6 mm diameter and 1 mm thickness. The clamped needles could be moved upwards and downwards at predetermined strain rates (velocities). During the puncture test, the clamped needle was brought down onto the hydrogel cylinders, which were placed on a stationary compression platen, at various strain rates (30 mm / min, 60 mm / min, 100 mm / min, or 200 mm / min). The test was carried out until 80% of the cylinder was penetrated and the corresponding load (N) was measured. The experiments were conducted with a minimum sample size of n > 5.Drag strength analysis:

[0082] To conduct the drag test analysis, hydrogels with dimension of 2 mm thickness and 6 mm diameter were casted with a surgical suture (10-0 Nylon suture) in the middle and cross-linked at an intensity of 31.06 mW / cm2. After the cross-linking step, the hydrogel samples with the nylon suture inside were immersed in IX PBS for 60 minutes for equilibrium swelling.

[0083] Subsequently, the hydrogel samples were placed on a compression platen of the BiSS uniaxial mechanical tester using a makeshift holder to prevent movement of the hydrogel sample during the testing process. The long end of the suture protruding from the hydrogel sample was attached to a wedge action grip positioned vertically above the sample.

[0084] During the test, the attached suture was pulled upward at a drag rate of 10 mm / min until it escaped from the hydrogel, and the corresponding load (N) was measured. The experiments were conducted with a minimum sample size of n > 5.

[0085] From the drag test experiment, drag strength and drag co-efficient was calculated using the formulae below:

[0086] Drag strength (kPa) = (Max. Load (N) / Surface area of the hollow cylinder in the hydrogel created by the movement of the suture (mm2)) x 1000.

[0087] Drag coefficient, pd= (FA-FB) / (FA+FB), where FA represents average maximum force, and FB represents average minimum force required to pull the suture fully out of the hydrogel.Results3D model of the printed lenticule:

[0088] Figure 2 shows the CAD model of the corneal lenticule with its proposed dimensions as per native cornea. The full length corneal lenticule was printed using the DLP-based bioprinter using the HA-MA / Gel-MA bioinks. The average print time for a full length lenticule was approximately 15 minutes. Isaacson and colleagues (Isaacson A, Swioklo S, Connon CJ. 3D bioprinting of a corneal stroma equivalent. Exp Eye Res. 2018;173:188-93. doi: 10.1016 / j.exer.2018.05.010) utilized a 3D printing technique to produce a curved structure resembling a cornea. However, thesize of the printed structure was notably larger than an actual cornea, and the surface finish was not optimal. In contrast, Duarte Campos and co-workers (Duarte Campos DF, Rohde M, Ross M, Anvari P, Blaeser A, Vogt M, et al. Corneal bioprinting utilizing collagen-based bioinks and primary human keratocytes. J Biomed Mater Res A. 2019;107(9): 1945-53. doi: 10.1002 / jbm.a.36702) detailed the creation of a corneal replacement using collagen-based bioinks and primary human keratocytes through bioprinting. The process of printing a complete structure with a diameter of 20 mm and a thickness of 0.3 mm took approximately one hour. In comparison to these approaches, the lenticules of the present disclosure were individually designed corneal replacements that circumvent the above issues in prior art. They can precisely conform to the recipient's eyeball, possessing an appropriate size and surface smoothness for corneal substitution, all achieved through a significantly faster manufacturing process. Optimization of bioink and printing parameters

[0089] Gelatin was chosen as a primary component of the bioprinted corneal lenticule as it structurally and functionally mimics collagen which is a primary component of the extra cellular matrix of native cornea. Further methacrylation of gelatin helps in formation of network structure. Hyaluronic acid was chosen as a second component for the bioprinted lenticule as it has been reported to exert control over the biomechanical properties of the cornea. The combination of HA-MA and Gel-MA was found to successfully print a full length corneal lenticule with optimum swelling and mechanical properties. The combination of HA-MA: Gel-MA (Table 1) printed lenticules were with optimum thickness (37 layers of 100 pm), good-handling and non-turbid appearance. In addition, a post-curing step (blue light, 20 mW / cm2, 10 minutes) further aided in improving the mechanical properties of the bioprinted lenticule. Also increasing the projection power output from 27.86 mW / cm2to 31.06 mW / cm2for the layer-by-layer photoirradiation further helped in improving the structural integrity and mechanical strength of the corneal lenticule (Figure 3).Compressive Modulus Measurements:

[0090] The successful bioengineering of bioprinted corneal lenticules for medical applications heavily relies on optimizing their mechanical properties. It is essential for a lenticule’s mechanical strength to closely match that of the native tissue to ensure it can offer sufficient structural support for complete and effective healing (S. Ulag et al., “Recent developments and characterization techniques in 3D printing of corneal stroma tissue, ” Polym Adv Technol, vol. 32, no. 8, pp. 3287-3296, 2021 ). Achieving this requires careful consideration of various factors, such as the concentration of polymers, photo-initiators, and other print-related parameters, which directly influence the mechanical strength of the bioprinted structures. The cylindrical discs printed using the same parameters as the bioprinted lenticule recorded a compressive modulus as indicated in Table 4 below. The compressive modulus was a bit on the higher side than the required value i.e., between 100-300 kPa. However, this moderately high compressive modulus did not impact the other desired properties of the printed lenticule. The biocompatibility studies of the present disclosure confirmed that the cells were alive and proliferating in the bioprinted lenticule formulation (Figure 5). The optimized bioprinted formulation has a careful trade-off of the physio-chemical properties desired for a successful ocular implant.Swelling Degree Measurements:

[0091] The swelling degree of the hydrogels is governed by the hydrogel structure i.e., the cross-linking networks formed between the polymers. The chemical cross-links formed between HA-MA and Gel-MA during the bioprinting impart adequate mechanical strength to the hydrogel, which is also confirmed by the high compressive modulus. The mass swelling percentage for the hydrogels are provided in the Table 4 below. The low swelling degree as observed is favourable as implants for ocular regeneration are expected to swell minimally. The low swelling degree is expected to ensure optical clarity, proper tissue integration as high swelling often results in corneal edema and imbalanced ocular pressure. The low swelling degree may impact the release of therapeutics from the hydrogels, but since these gels are susceptible to enzymatic degradation, a sustained release is expected.Adhesion Strength Analysis:

[0092] The average adhesion strength for the bioprinted lenticule formulations / hydrogels are provided in Table 4 below (Figure 4(a) provides graph of working composition 1). It is anticipated that the lenticules in the wound site will effectively bond with the adjacent and underlying tissue. Therefore, a higher degree of adhesive potency to the tissue is desirable. In the present disclosure adhesion strength of the lenticules was higher than the reported values for clinically available sealants (Evicel: 19.4 ± 17.3 kPa) and CoSEAL: 26.3 ± 4.7 kPa) (E. Shirzaei Sani et al., “Sutureless repair of corneal injuries using naturally derived bioadhesive hydrogels, ” Sci Adv, vol. 5, no. 3, p. eaav!281, 2019). Also, it was higher than Quixil (24.6 kPa), Beriplast (24.2 kPa) but lower than other commercial sealants like Tachosil (59.6 kPa), and Tisseel (77.5 kPa).Puncture resistance analysis:

[0093] While designing hydrogels for corneal implantation, it is crucial to evaluate the potential damage that may occur during surgical suturing, if required. Although the lenticules of the present disclosure may not necessitate suturing, depending on in-vivo conditions, there might be situations where suturing is needed. Therefore, a puncture analysis test was conducted using a Ethilon 10-0 black monofilament surgical.

[0094] By comparing the puncture strength values at different insertion rates (30 mm / min, 60 mm / min, 100 mm / min, and 200 mm / min), there was observed a consistent decrease in puncture strength with increasing insertion rates (Figure 4d for working composition 1). The puncture strength for the working composition 1 was noted to be 130.63±11.58.

[0095] It is important to note that irrespective of different insertion rates, there was no observed substantial damage to the overall structure of the hydrogels. This suggests that the hydrogels possess inherent resistance to puncture damage. Further, puncture strength properties can also be a measure of toughness indicating resistance to fracture or break (G. W. Radebaugh, J. L. Murtha, T. N. Julian, and J. N. Bondi, “Methods forevaluating the puncture and shear properties of pharmaceutical polymeric films, ” Int J Pharm, vol. 45, no. 1—2, pp. 39—46, 1988).

[0096] Overall, these findings indicate that the hydrogels exhibit notable resistance to puncture damage, which is an important characteristic for their potential application in corneal implantation procedures.Drag strength analysis:

[0097] While the puncture strength analysis provides insight into the resistance to needle-induced damage during insertion, it is equally important to assess the damage caused to the hydrogel when a surgical suture is dragged across its cross-sectional area during suturing. To investigate this aspect, a drag strength analysis was conducted using a nylon surgical suture dragged across the hydrogel.

[0098] The average drag strength exerted on the lenticule, with a drag rate of 10 mm / min for the surgical suture, is indicated in Table 4 below (Figure 4f provides graph for working composition 1). The puncture strength for the working composition 1 was noted to be 19.26±3.87.

[0099] Like the observations in the puncture test analysis, no significant damage to the lenticule structure was observed due to the dragging of the suture. This suggests a potential surgical suitability of the lenticules.

[0100] Table 3: indicating maximum ascending and minimum descending force applied on the hydrogel as a result of dragging the surgical suture across its cross - section.Table 4: Result for Working compositions of Table 1

[0101] Table 5: Result for the Non-working compositions of Table 2EXAMPLE 2In vitro biocompatibilitv assessment of the hydrogelCell encapsulation in hydrogel

[0102] The bioink (working composition 1) was encapsulated with 3rd passage human corneal stromal cells (hCSCs, procured commercially) at a density of 5 x 106cells / mL of hydrogel. The cell suspension was mixed with the bioink solution and 20 pL was dropped on to a sterile glass slide. This was then photo-crosslinked using blue light of 31.06 mW / cm2for 1 minute corresponding to the LumenX printing parameters. Thereafter, the hCSCs encapsulated hydrogels were submerged in the complete media and incubated at 37°C, 5% CO2, along with media change on every 3rd day.Cell viability assessment

[0103] At specified intervals of Day 1, 7, 14, and 21, the hydrogels containing hCSCs were taken out from the culture. To evaluate cell viability, a viability assessment medium was used, consisting of calcein acetoxymethyl (calcein-AM) at a concentration of 0.2 pg / mL and ethidium homodimer (EthD-1) at a concentration of 2.5 pg / mL (obtained from Invitrogen, Paisley, UK). The viability assessment medium was prepared in MEM media, and it was applied to the samples, followed by an incubation period of 15 minutes at 37°C. Subsequently, the samples were examined using a confocal microscope (Stellaris, Leica microsystems) to visualize the distribution of green, fluorescent signals from live cells and red stained signals indicating dead cells. Biomarker assessment:

[0104] The hydrogels containing hCSCs, collected at different time points, underwent a series of procedures. They were first washed with PBS and then fixed using a 10% formalin solution (Sigma-Aldrich, India) for 20 minutes at room temperature. Afterward, the samples were washed with IX PBS and blocked using a 5% BSAsolution in IX PBS for 45 minutes. For immunostaining, primary antibodies against CD90 and alpha smooth muscle actin (a-SMA), obtained from Abeam, were used at a 1:100 dilution in 1% BSA. The samples were incubated with the primary antibodies overnight at 4°C. After removing the primary stains, the samples were washed with IX PBS and then exposed to secondary antibodies (goat anti-mouse and anti-rabbit antibodies conjugated with Alexa Fluor 594 and Alexa Fluor 488 from Thermo Fisher Scientific) at a 1:200 dilution in 1% BSA. The incubation with secondary antibodies took place at room temperature for 1 hour. Following the incubation with secondary antibodies, any unbound antibodies were washed away using IX PBS. To visualize the cell nuclei, Vectashield antifade mounting medium containing DAPI (#H1200, Vector Labs, Burlingame, CA, USA) was mounted on top of the sample. Finally, the samples were examined using a confocal microscope (Stellaris, Leica microsystems) to identify the cell phenotype within the hydrogels.In vitro Re-epithelialization studies:

[0105] To assess the re-epithelialization potential of the lenticule, human corneal epithelial cells were seeded on top of the lenticules. The seeding density for these hydrogels was 7x104cells / cm2Epithelial cells were seeded at a density of 1x104cells / cm2onto collagen coated coverslips to make 2D Control samples. Phase -contrast images along with immunofluorescence staining images for ZO-1 (1:50, anti-ZOl raised in Rabbit, Invitrogen, 61-7300) and Rhodamine Phalloidin (Thermofisher scientific R415) were captured using a confocal microscope (Stellaris, Leica microsystems) when the cells reached confluency.ResultsCytocompatibility of HA-MA / GeLMA hydrogels

[0106] To assess the biocompatibility of the moulded corneal lenticules, human corneal stromal cells (hCSCs) were encapsulated. The cell viability study demonstrated viability of > 95%, indicating that the lenticule was cytocompatible, with minimal cell death observed in later time points as shown in Figure 5. Figure 5 depicts the viability of human corneal stromal cells evaluated on both 2D surfaces (a & b) and within 3Dbioprinted lenticules (c-f) to observe the changes in viable cell population over a span of 3 weeks. The z-axis projection (g-j) of the frames captured for the lenticules revealed a uniform distribution of viable cells. In the visual representation, viable cells appear green, while cell nuclei are blue, and any dead cells appear red. The cells exhibited normal morphology and proliferation profiles over the period of culture. Thereafter, immunofluorescence staining was performed to assess the phenotype of the encapsulated cells, wherein CD90 enacted as a sternness marker and aSMA demarcated the fibrotic cells. CD90 is considered to be the gold standard marker for mesenchymal stem cells (MSCs) (F.-.J. Lv, R. S. Tuan, K. M. C. Cheung, and V. Y. L. Leung, “Concise Review: The Surface Markers and Identity of Human Mesenchymal Stem Cells, ” Stem Cells, vol. 32, no. 6, pp. 1408—1419, Jun. 2014, doi: 10.1002 / stem.l681). Also, previous studies have shown limbal mesenchymal stromal cells to exhibit > 90% CD90 expression (L. J. Bray, C. F. Heazlewood, K. Atkinson, D. W. Hutmacher, and D. G. Harkin, “Evaluation of methods for cultivating limbal mesenchymal stromal cells, ” Cytotherapy, vol. 14, no. 8, pp. 936—947, Sep. 2012, doi: 10.3109 / 14653249.2012.684379).

[0107] Figure 6 depicts the results of cells subjected to phenotype evaluation through immunofluorescence staining after being in culture for 3 weeks. For hCSCs cultured on a coverslip (a-b), and those encapsulated within 3D bioprinted lenticules (c-f), the staining was performed for CD90 (red), the sternness marker, and a-SMA (green), a fibrosis marker. The cell nucleus was stained blue. This figure shows that the encapsulated cells were stained positively for CD90 depicting their sternness state. The fibrosis marker, a-SMA is a commonly utilized indicator for identifying activated fibrocytes / myofibroblasts, which have a significant impact on tissue fibrosis (R. C. de Oliveira and S. E. Wilson, “Fibrocytes, Wound Healing, and Corneal Fibrosis, ” Investigative Opthalmology & Visual Science, vol. 61, no. 2, p. 28, Feb. 2020, doi: 10.1167 / iovs.61.2.28). aSMA expression was minimal among the encapsulated cells in the hydrogel formulation in all the time points showing that there was minimal transition of the CSCs to fibrotic state overtime.

[0108] Rapid re-epithelialization is crucial to guard against infections and promote successful implant-host integration. Previous in vitro research has shown that the mechanics and surface roughness of biomaterials play a significant role in influencing the migration and maturation of epithelial cells (R. M. Gouveia, G. Lepert, S. Gupta, R. R. Mohan, C. Paterson, and C. J. Connon, “Assessment of corneal substrate biomechanics and its effect on epithelial stem cell maintenance and differentiation, ” Nat Commun, vol. 10, no. 1, p. 1496, Apr. 2019, doi: 10.1038 / s41467-019-09331-6). Hence, in the bioprinted lenticules, it was verified whether the seeded epithelial were altered under the influence of the bioink components. Phase contrast images from Figure 7 revealed that the epithelial cells exhibited healthy morphology when cultured on the surface of the lenticule. The cells were also stained for evaluating the expression of ZO1 proteins and Rhodamine-phalloidin. Previously, it has been demonstrated that ZO proteins undergo nuclear shuttling (M. Benezra, R. S. Greenberg, and S. K. Masur, “Localization of ZO-1 in the Nucleolus of Corneal Fibroblasts, ” Investigative Opthalmology & Visual Science, vol. 48, no. 5, p. 2043, May 2007, doi: 10.1167 / iovs.06-0754). In addition to their protein domains, ZO proteins also possess specific Nuclear Localization Signals (NLS) and Nuclear Export Signals (NES) that facilitate their movement between the nucleus and cytoplasm. Before the tight junctions mature, ZO1 is observed to stain the nucleus of sub-confluent epithelial cells. The IF images from Figure 7 showed positive expression of ZO1 proving that the seeded cells were maintaining healthy phenotype. Even F-actin was successfully stained (in red) by rhodamine-phalloidin dye demonstrating that our bioprinted lenticule actively supports the growth of epithelial cells.Advantages of the present disclosure

[0109] The present disclosure provides a three-dimensional hydrogel or corneal lenticule with the following advantages: a) The hydrogel is having transmittance in a range of 80 to 94% ; mass swelling in a range of 1 to 45%; a compressive modulus in a range of 400 to 600 kPa; andan adhesion strength of 30 to 80 kPa, which all closely mimics the properties of natural cornea. b) The lenticule of the present disclosure is prepared by a robust DLP technology with accurate shape parameters. c) The lenticule exhibits biocompatibility.

Claims

I / We claim:

1. A three-dimensional hydrogel comprising a plurality of layers forming a three- dimensional structure, wherein each layer is a polymeric scaffold comprising cross-liked methacrylated hyaluronic acid and methacrylated gelatin.

2. The three-dimensional hydrogel as claimed in claim 1, wherein the polymeric scaffold is obtained using a bio-ink composition comprising, in an aqueous carrier: a) the methacrylated hyaluronic acid; b) the methacrylated gelatin; c) a photoinitiator; and d) a photoabsorber.

3. The three-dimensional hydrogel as claimed in claim 2, wherein the methacrylated hyaluronic acid is in a weight percentage range of 1.81 to 6.35% (20-70 mg / ml); and methacrylated gelatin is in a weight percentage range of 1.81 to 10.88% (20-120 mg / ml), in respect of the bioink composition.

4. The three-dimensional hydrogel as claimed in claim 2, wherein the degree of substitution of methacrylate groups in the methacrylated hyaluronic acid is in a range of 20 to 75%; and the degree of substitution of methacrylate groups in the methacrylated gelatin is in a range of 40 to 99%.

5. The three-dimensional hydrogel as claimed in claim 2, wherein the molecular weight of hyaluronic acid in the methacrylated hyaluronic acid is in a range of 5 to 60 kDa; and the molecular weight of gelatin in the methacrylated gelatin is in a range of 50 to 1000 kDa.

6. The three-dimensional hydrogel as claimed in claim 2, wherein the bio-ink composition further comprises a plurality of cells, drugs, extracellular vesicles, nanovesicles, or mixtures thereof.

7. The three-dimensional hydrogel as claimed in claim 2, wherein the photoinitiator is selected from sodium persulphate (SPS), ruthenium, lithiumphenyl-2,4,6-trimethylbenzoylphosphinate (LAP), riboflavin, 2-Hydroxy-4'- (2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959), or mixtures thereof.

8. The three-dimensional hydrogel as claimed in claim 2, wherein the photoinitiator is in a weight percentage range of 0.005 to 0.7% (1 - 30 mM), in respect of the bioink composition.

9. The three-dimensional hydrogel as claimed in claim 2, wherein the photoabsorber is selected from tartrazine, fast green FCF, new coccine, methylene blue, or mixtures thereof.

10. The three-dimensional hydrogel as claimed in claim 2, wherein the photoabsorber is in a weight percentage range of 0.007 to 0.04% (0.1635 mM - 0.754 mM) in respect of the bioink composition.

11. The three-dimensional hydrogel as claimed in claim 2, wherein the aqueous carrier is selected from phosphate buffered saline, saline, water, or mixtures thereof.

12. The three-dimensional hydrogel as claimed in claim 2, wherein the aqueous carrier is in a weight percentage range of 70 to 95% in respect of the bioink composition.

13. The three-dimensional hydrogel as claimed in claims 1-2, wherein the three- dimensional hydrogel encapsulates a plurality of cells, drugs, extracellular vesicles, nanovesicles, or mixtures thereof.

14. The three-dimensional hydrogel as claimed in claims 1-2, wherein the hydrogel is characterized by transmittance in a range of 80 to 94% ; mass swelling in a range of 1 to 45%; a compressive modulus in a range of 400 to 600 kPa; and an adhesion strength of 30 to 80 kPa.

15. A method for preparing the three-dimensional hydrogel as claimed in claims 1- 2, the process comprising: preparing a bioink composition comprising methacrylated hyaluronic acid, methacrylated gelatin, photoinitiator, and photoabsorber in an aqueous carrier; and fabricating the three-dimensionalhydrogel, layer-by-layer, wherein each layer is separately exposed to photoirradiation for cross-linking of bioink.

16. The method as claimed in claim 15, wherein the layer-by-layer fabrication is performed by a digital light processing printer.

17. The method as claimed in claim 16, wherein the shape and dimensions of the three-dimensional hydrogel is based on a quantitative model defining a desired shape and dimensions of the lenticules to match a recipient.

18. The method as claimed in claim 15, wherein the photoirradiation is carried out using light of intensity in the range of 10 to 35 mW / cm219. The method as claimed in claim 15, wherein the photoirradiation is carried out for a time period in the range of 0.2 to 30 seconds.

20. The method as claimed in claim 15, wherein the photoirradiation is carried out at a wavelength in the range of 400 to 700nm, preferably in the range of 400 to 450nm, more preferably in the range of 400 to 415nm.

21. The method as claimed in claim 15, wherein the method comprises an optional step of: removing excess non-crosslinked bioink, and post curing the three- dimensional hydrogel in the presence of light of 380 to 450 nm wavelength.

22. The method as claimed in claim 21 , wherein the post-curing is for a time period in the range of 5 minutes to 20 minutes.

23. The method as claimed in claim 15, wherein the method comprises providing a plurality of cells, drugs, extracellular vesicles, nanovesicles or mixtures thereof, to the bioink composition prior to fabricating the three-dimensional hydrogel.

24. The method as claimed in claim 15, wherein the three-dimensional hydrogel is in the form of a lenticule.

25. A method of treating a corneal tissue repair in need thereof, comprising implanting the three-dimensional hydrogel as claimed in claim 1 , in the corneal tissue.

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