Collagen gel formulation

Abstract

The present inventors have developed a printable collagen bio-ink using unmodified type I collagen that has mechanical and structural properties that facilitate application in a structured form to a tissue. In particular, the compositions of the present invention can be used to apply collagen gels to a tissue (e.g., an eye) using two-dimensional or three-dimensional (extrusion) bioprinting techniques.

Description

[0001] Incorporating via cross-references

[0002] This application claims priority to Australian Provisional Patent Application No. 2019904331, filed on November 18, 2019, the entire contents of which are incorporated herein by cross-reference. Technical Field

[0003] This invention generally relates to the fields of biology and medicine. More specifically, this invention relates to compositions suitable for delivering pharmaceutical agents to biological targets such as tissues and cells and / or providing protection to said biological targets, and methods for producing the same.

[0004] background

[0005] When biological tissue is damaged, for example due to accidental mechanical injury, surgery, or disease, the primary task is to effectively seal the damaged site. Tissue sealants have a wide range of applications, including but not limited to preventing further damage, preventing infection, and minimizing blood loss. Increasingly, tissue sealants are also being used to deliver medications to damaged tissue, which may aid healing and / or help prevent infection. When sealants are made from natural materials, the likelihood of adverse reactions such as allergic reactions is lower.

[0006] The mechanisms of action and ease of use of occlusives vary greatly. Effective occlusives flow freely during application but retain their shape / structure afterward. This latter property is especially important when the shape of the biological tissue being repaired (e.g., corneal tissue) is crucial to its function.

[0007] The cornea is a transparent component that protects the eye. It allows light to pass through the pupil and is the primary refractive element of the eye's optical system. The cornea consists of five layers: the outer epithelium, the anterior elastic lamina, the stroma, the des elastic lamina, and the endothelium. The corneal stroma accounts for approximately 90% of the total thickness of the cornea and is primarily composed of collagen.

[0008] Corneal blindness is the leading cause of blindness worldwide, second only to cataracts in terms of the number of cases. The causes of corneal blindness are varied, including diseases such as trachoma, onchocerciasis, leprosy, neonatal ophthalmia, and dry eye syndrome, as well as other processes such as eye trauma, corneal ulcers, and complications from the use of traditional eye drops.

[0009] Corneal injury is the most common ophthalmic emergency in Australia, accounting for approximately 75% of cases due to foreign bodies or abrasions in the cornea. It is estimated that these injuries alone cost the Australian population over US$155 million annually, and if left untreated, can lead to infection and scarring, resulting in permanent vision loss.

[0010] In mild cases, a damaged cornea can regenerate through normal healing processes. However, in other cases, the cornea's normal healing mechanisms are insufficient, leading to non-healable defects that can cause corneal melting, corneal neovascularization, loss of transparency, infection, scarring, and decreased vision, even blindness.

[0011] Current medical treatments for corneal injuries include antibiotics, eye pads, sutures, and surgical adhesive tape, which may help resolve minor issues. However, they do not adequately address problems that arise in more advanced cases, including pain relief, infection, and / or scar tissue formation. Infection is a serious complication that often requires hospitalization. Scarring is common in severe corneal injuries and can lead to permanent vision loss. In such cases, corneal transplantation is the only option for vision rehabilitation, but there is a global shortage of donor corneas.

[0012] Many of the problems mentioned above are not limited to corneal damage; they are also common in cases of damage to other body tissues.

[0013] There is a need for improved compositions and methods for sealing biological tissues and / or for effectively delivering agents to biological targets such as tissues and cells. Invention Overview

[0015] This invention alleviates at least one problem associated with current compositions and / or methods for sealing biological tissues and delivering agents to biological targets. In the case of the cornea, one of the main current methods is to reconstruct corneal defects using natural or synthetic materials. Collagen is the primary natural material used and is the main protein in the cornea. Collagen accounts for 60%–80% of the dry weight of the cornea, and type I collagen is the dominant type in the corneal stroma. However, the strength and elasticity of currently produced naturally occurring collagen products are limited. Chemical modification or cross-linking is usually required to improve the robustness of type I collagen and other types of collagen generated for therapeutic purposes. The biomechanical properties of current collagen-based products are still insufficient for sustainable use, as irregular corneal shapes are often observed over time.

[0016] In addition to collagen implants, collagen occlusives and bio-inks have been proposed and developed. However, the application of such bio-inks is limited by complex printing methods and the lack of transparency in the resulting products.

[0017] Instead of using collagen implants as a matrix equivalent, the inventors have developed a printable collagen bio-ink that uses unmodified type I collagen to be directly printed onto the cornea. The bio-ink of this invention can also be directly printed onto a range of other biological targets, such as tissues, membranes, and cells. The printed collagen bio-ink can solidify to form a temporary structure that is safely degraded and allows for the migration of surrounding corneal cells and matrix remodeling. The collagen bio-ink of this invention can be printed using two-dimensional or three-dimensional (extrusion) bioprinting techniques. Furthermore, the bio-ink of this invention is capable of photocrosslinking. The bio-ink described herein can be transparent and viscous, and can support cell migration and proliferation. Biodegradable bio-inks can be used to deliver bioactive molecules, such as growth factors.

[0018] In non-limiting terms, the compositions and methods described herein can generally be used to deliver agents (e.g., drugs and / or other substances) to biological targets (e.g., tissues, membranes, cells) and can be applied to, for example, closed tissues, including corneal tissue.

[0019] This invention relates in at least part to the following embodiments:

[0020] Implementation Plan 1 A composition comprising:

[0021] Type I collagen: -3-15 mg / ml;

[0022] Sodium ions of -0.135–0.5 M and / or calcium ions of 0.008–0.4 M; and

[0023] - One or more cross-linking agents.

[0024] Implementation Plan 2 The composition according to embodiment 1, wherein the composition contains 0.01-0.5% (w / v) riboflavin or 0.01-0.5% (w / v) rose red.

[0025] Implementation Plan 3. The composition according to embodiment 1 or embodiment 2, wherein the one or more crosslinking agents can be photoactivated.

[0026] Implementation Plan 4 The composition according to embodiment 3, wherein the light is UV light, blue light, green light or white light.

[0027] Implementation Plan 5 The composition according to embodiment 1, wherein the composition comprises fibrinogen and / or thrombin.

[0028] Implementation Plan 6 The composition according to embodiment 5, wherein the composition comprises 1.6-6 mg / ml of fibrinogen.

[0029] Implementation Plan 7 The composition according to embodiment 5 or embodiment 6, wherein the composition contains 1-5 U / mL of thrombin.

[0030] Implementation Plan 8 The composition according to any one of embodiments 1 to 7 further comprises one or more of the following: culture medium, growth factor, hormone, matrix protein, glycoprotein, vitamin, ion other than sodium or calcium ions, ion source, fibronectin, amino acid, antibiotic, anesthetic, factor XIII, fetal bovine serum (FBS), human serum, platelet lysate, and human platelet lysate.

[0031] Implementation Plan 9. The composition according to embodiment 8, wherein the composition comprises a culture medium containing ions and amino acids.

[0032] Implementation Plan 10 The composition according to embodiment 8 or embodiment 9, wherein:

[0033] (i) The growth factors include human epidermal growth factor (hEGF) and / or fibroblast growth factor (FGF); and / or

[0034] (ii) Vitamins include ascorbate (vitamin C); and / or

[0035] (iii) Matrix proteins include type IV collagen; and / or

[0036] (iv) Hormones include insulin; and / or

[0037] (v) Glycoproteins include transferrin.

[0038] Implementation Plan 11. The composition according to any one of embodiments 1 to 10, wherein the ion is a component of the ionic salt included in the composition.

[0039] Implementation Plan 12 The composition according to any one of embodiments 1 to 11, wherein the composition further comprises mammalian cells.

[0040] Implementation Plan 13 The composition according to embodiment 12, wherein the mammalian cells comprise or are composed of human cells.

[0041] Implementation Plan 14 The composition according to any one of claims 1 to 13, wherein type I collagen is neutral.

[0042] Implementation Plan 15The composition according to any one of embodiments 1 to 14, wherein the composition comprises:

[0043] (i) 3-15 mg / ml of type I collagen, 0.135-0.2 M of sodium ions, and 0.01-0.05 M of calcium ions; or

[0044] (ii) 4-12 mg / ml of type I collagen, 0.135-0.16 M of sodium ions, and 0.015-0.03 M of calcium ions; or

[0045] (iii) 5-11 mg / ml of type I collagen, 0.135-0.14 M of sodium ions and 0.018-0.02 M of calcium ions.

[0046] Implementation Plan 16 The composition according to any one of embodiments 1 to 15, wherein the composition comprises:

[0047] (i) Type I collagen less than 15 mg / ml;

[0048] (ii) Sodium ions exceeding 0.135 M; and

[0049] (iii) Calcium ions exceeding 0.018 M.

[0050] Implementation Plan 17 A method for preparing a composition, the method comprising:

[0051] (i) Provide a solution comprising:

[0052] Type I collagen: -3-15 mg / ml;

[0053] - One or more crosslinking agents; and

[0054] -0.135-0.5M sodium ions and / or 0.008-0.4M calcium ions;

[0055] (ii) Apply the solution to the surface; and

[0056] (iii) Apply light to the solution that can activate one or more crosslinking agents.

[0057] Implementation Plan 18 According to the method of embodiment 17, the solution is applied to form a surface layer, and steps (ii) and (iii) are repeated multiple times, wherein each layer is applied over the previous layer.

[0058] Implementation Plan 19The method according to embodiment 17 or embodiment 18, wherein the one or more crosslinking agents contain 0.01-0.5% (w / v) riboflavin or 0.01-0.5% (w / v) rose red, and wherein the light includes UV light, blue light, green light or white light.

[0059] Implementation Plan 20 The method according to any one of embodiments 17 to 19, wherein:

[0060] (a) Combining one or more crosslinking agents with a base to form part A;

[0061] (b) Combining type I collagen with sodium and / or calcium ions to form part B; and

[0062] (c) Prior to step (iii), mix part A and part B to form a solution.

[0063] Implementation Plan 21 A method for preparing a composition, the method comprising:

[0064] (i) Provide a solution comprising:

[0065] Type I collagen: -3-15 mg / ml;

[0066] - One or more crosslinking agents; and

[0067] -0.135-0.5M sodium ions and / or 0.008-0.4M calcium ions;

[0068] (ii) Applying the solution to a surface, wherein the solution is divided into at least two components before being applied to the surface.

[0069] Implementation Plan 22 The method according to embodiment 21 further includes the following steps:

[0070] (iii) Adding fibrinogen to at least one component to form a formulation (a);

[0071] (iv) Adding thrombin to at least one component to form preparation (b); and

[0072] (v) Combine formulations (a) and (b) to form a gel.

[0073] Implementation Plan 23 According to the method described in implementation scheme 22, wherein:

[0074] - In step (iii), 1.6-6 mg / ml fibrinogen is added to at least one component to form formulation (a); and / or

[0075] - In step (iv), 1.5 U / mL thrombin is added to at least one component to form preparation (b).

[0076] Implementation Plan 24 According to any one of embodiments 17 to 23, the solution further comprises one or more of the following: culture medium, growth factor, hormone, matrix protein, glycoprotein, vitamin, ions other than sodium or calcium ions, ion source, fibronectin, amino acid, antibiotic, anesthetic, factor XIII, fetal bovine serum (FBS), human serum, platelet lysate, and human platelet lysate.

[0077] Implementation Plan 25 The method according to embodiment 24, wherein the solution contains a culture medium containing ions and amino acids.

[0078] Implementation Plan 26 The method according to implementation scheme 24 or implementation scheme 25, wherein:

[0079] (i) The growth factors include human epidermal growth factor (hEGF) and / or fibroblast growth factor (FGF); and / or

[0080] (ii) Vitamins include ascorbate (vitamin C); and / or

[0081] (iii) Matrix proteins include type IV collagen; and / or

[0082] (iv) Hormones include insulin; and / or

[0083] (v) Glycoproteins include transferrin.

[0084] Implementation Plan 27 The method according to any one of embodiments 17 to 26, wherein the ion is a component of an ionic salt included in the solution.

[0085] Implementation Plan 28 The method according to any one of embodiments 17 to 27, wherein the solution further comprises mammalian cells.

[0086] Implementation Plan 29 The method according to embodiment 28, wherein the mammalian cell comprises or is composed of human cells.

[0087] Implementation Plan 30. The method according to any one of embodiments 17 to 29, wherein type I collagen is neutral.

[0088] Implementation Plan 31A composition obtained by or capable of being obtained by the method according to any one of embodiments 17 to 30.

[0089] Implementation Plan 32 A method for sealing a tissue surface, the method comprising applying the composition according to any one of embodiments 1 to 16 or 31 to the tissue.

[0090] Implementation Plan 33 A method of delivering a drug agent to a tissue, the method comprising applying the composition according to any one of embodiments 1 to 16 or 31 to the tissue.

[0091] Implementation Plan 34 The composition according to any one of embodiments 1 to 16 or embodiment 31 is used to seal the tissue surface.

[0092] Implementation Plan 35 The composition according to any one of embodiments 1 to 16 or embodiment 31 is used for delivering a pharmaceutical agent to a tissue.

[0093] Implementation Plan 36 Use of a kit, package, or apparatus comprising type I collagen, sodium ions, calcium ions, and one or more cross-linking agents for the preparation of a composition comprising:

[0094] Type I collagen: -3-15 mg / ml;

[0095] Sodium ions of -0.135–0.5 M and / or calcium ions of 0.008–0.4 M; and

[0096] - One or more cross-linking agents.

[0097] Implementation Plan 37 For use in the kit, packaging, or device according to embodiment 36, wherein the composition comprises 0.01-0.5% (w / v) riboflavin or 0.01-0.5% (w / v) rose sulfide.

[0098] Implementation Plan 38 According to the intended use of the kit, packaging, or device described in embodiment 36 or 37, wherein the one or more crosslinking agents are photoactivated.

[0099] Implementation Plan 39 The intended use of the reagent kit, packaging, or device according to embodiment 38, wherein the light is UV light, blue light, green light, or white light.

[0100] Implementation Plan 40 According to the use of the kit, packaging, or device described in embodiment 36, wherein the composition comprises fibrinogen and thrombin, and wherein...

[0101] - Fibrinogen is present in the first compartment;

[0102] - Thrombin is present in the second compartment; and in which

[0103] - The kit, package, or device is configured to allow the separation of fibrinogen in the first compartment and thrombin in the second compartment during and after loading fibrinogen and thrombin into the kit, package, or device, and wherein the kit, package, or device further includes a tool for facilitating the mixing of fibrinogen in the first compartment with thrombin in the second compartment.

[0104] Implementation Plan 41 According to the use of the reagent kit, packaging, or device as described in embodiment 40, wherein the composition comprises:

[0105] -1.6-6 mg / ml of fibrinogen; and / or

[0106] Thrombin concentration: -1-5 U / mL

[0107] Implementation Plan 42 The use of the kit, packaging, or device according to any one of embodiments 36 to 41, wherein the composition further comprises one or more of the following: culture medium, growth factor, hormone, matrix protein, glycoprotein, vitamin, ion other than sodium or calcium ions, ion source, fibronectin, amino acid, antibiotic, anesthetic, factor XIII, fetal bovine serum (FBS), human serum, platelet lysate, and human platelet lysate.

[0108] Implementation Plan 43. According to any one of embodiments 36 to 42, the kit, packaging or device is used in a manner in which any one or more of type I collagen, sodium ions, calcium ions and / or one or more cross-linking agents are separated from one or more other components within the kit.

[0109] Implementation Plan 44 The composition according to any one of embodiments 1 to 16 or embodiment 31, wherein the composition is transparent.

[0110] Implementation Plan 45 The composition according to any one of embodiments 1 to 16 or embodiment 31, wherein the composition has the ability to retain or substantially retain its shape / structure after printing.

[0111] definition

[0112] As used in this application, the singular forms “a,” “an,” and “the” include plural references unless the context clearly specifies otherwise. For example, the term “component” also includes multiple components.

[0113] As used herein, the term “comprising” means “including”. Variations of the word “comprising”, such as “comprise” and “comprise”, have various corresponding meanings. Thus, for example, a composition that “comprising” component “A” may consist of only component “A” or may include one or more additional components (e.g., component “B” and / or component “C”).

[0114] As used herein, the term "subject" includes any animal of economic, social, or research importance, including cattle, horses, sheep, primates, birds, and rodent species. Therefore, "subject" can be a mammal, such as a human or a non-human mammal.

[0115] As used in this article, the term “tissue” will be understood to encompass both cells, which are components of tissue, and organs formed from tissue.

[0116] As used herein, the term “kit” refers to any delivery system for delivering materials. Such delivery systems include systems that allow the storage, transport, or delivery of reaction reagents (e.g., labels in appropriate containers, reference samples, support materials, etc.) and / or support materials (e.g., buffer solutions, written instructions for performing the assay, etc.) from one location to another. For example, a kit may include one or more housings (such as boxes) containing the relevant reaction reagents and / or support materials. The term “kit” includes segmented kits and combination kits. A “segmented kit” refers to a delivery system containing two or more separate containers, each containing a sub-part of all kit components. The containers may be delivered together or individually to the intended subject. Any delivery system containing two or more separate containers is included within the meaning of the term “segmented kit,” where each container contains a sub-part of all kit components. A “combination kit” refers to a delivery system containing all components of the reaction assay in a single container (e.g., in a single box containing each of the desired components).

[0117] As used herein, the term “about” when referring to the listed values ​​includes both the listed values ​​and values ​​within a range of 10% plus or minus the listed values.

[0118] As used herein, the term "multiple" means more than one. In certain aspects or implementations, multiple can mean 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 or more, as well as any numerical value and any range that can be derived therefrom.

[0119] As used herein, the term "between" encompasses the values ​​at each endpoint of a numerical range when used to refer to such a range. For example, sodium ions with concentrations between 0.135 M and 3 M include sodium ions with concentrations of 0.135 M and 3 M.

[0120] As used herein, the term “greater than” will be understood as “greater than or equal to” when used to refer to numerical values. For example, sodium ions greater than 0.135 M encompass sodium ion concentrations of 0.135 M and all sodium ion concentrations greater than 0.135 M.

[0121] As used herein, the term “less than” will be understood as “less than or equal to” when referring to numerical values. For example, type I collagen less than 15 mg / ml encompasses type I collagen at a concentration of 15 mg / ml and all concentrations of type I collagen less than 15 mg / ml.

[0122] As used herein, the term “neutral” when used to describe type I collagen will be understood to mean that the pH of the collagen solution is between 6.7 and 7.6. For example, “neutral” type I collagen may have the following pH values: between 6.8 and 7.5, or between 6.9 and 7.4, or between 7.0 and 7.3, or between 6.9 and 7.2, etc.

[0123] Any description of prior art documents, or statements derived from or based on those documents, herein does not imply that those documents or derived statements are part of the common general knowledge of the relevant art.

[0124] For descriptive purposes, all documents mentioned herein are incorporated herein by reference in their entirety unless otherwise stated. Attached Figure Description

[0125] Preferred embodiments of the invention will now be described by way of example only with reference to the accompanying drawings, in which:

[0126] Figure 1The results of testing the effect of ions on the stability of neutral type I collagen solutions are shown. For each solution generated, 90 parts of collagen solution were neutralized with 2.7 parts of 5M NaOH and then mixed with 7.3 parts of various salt solutions. The solutions tested included 1× phosphate-buffered saline (PBS, composed of NaCl, KCl, Na2HPO4, and KH2PO4), NaCl, KCl, Na2HPO4, KH2PO4, and sodium ascorbate (NaC6H7NO6). The solutions were left on the workbench for 1 hour, 1 day, and 1 week before examining precipitation formation. The composition of 1×PBS (mM) is NaCl, KCl, Na2HPO4, KH2PO4, and sodium ascorbate (NaC6H7NO6). + 156.9, K + 109.8, Cl - 244.9, HPO4 2- 10. H2PO 4- :1.8. Figure 1 It was shown that either NaCl (minimum concentration 68.4 mM) or CaCl2 (minimum concentration 18 mM) is optimal for constructing soluble transparent collagen gels.

[0127] Figure 2 Riboflavin powder was dissolved in CaCl2 / PBS. Figure 2 a shows riboflavin in CaCl2 before centrifugation. Left tube: 0.1% riboflavin solution; Right tube: 0.2% riboflavin solution. Figure 2 b shows riboflavin in CaCl2 after centrifugation. Left tube: 0.1% riboflavin solution remains clear. Right tube: 0.2% riboflavin precipitates in CaCl2 solution, as indicated by the arrow.

[0128] Figure 3 Representative images of cross-linked collagen ink on a glass slide are provided. Figure 3 a shows collagen ink crosslinked by a tissue culture hood UV lamp. Figure 23 It shows 3mw / cm 2 365nm UV crosslinked collagen ink. Figure 3 c shows the output of 470nm 10mw / cm 2 Blue light crosslinked collagen ink.

[0129] Figure 4 Provided by 3mw / cm 2 Representative image of human collagen-based collagen ink crosslinked with 365nm UV on a glass slide.

[0130] Figure 5 Representative images of collagen bio-inks crosslinked with rose red-green light are provided.

[0131] Figure 6These are a series of graphs showcasing the photorheological properties of collagen bio-ink samples. Figure 6 a to Figure 6 e shows the changes in storage modulus and loss modulus over time after UV light is applied to the sample. Figure 6 a shows 12 mg / ml collagen containing PBS. Figure 6 b shows 12 mg / ml collagen containing calcium ions. Figure 6 c shows 6 mg / ml collagen containing calcium ions. Figure 6 d shows 3 mg / ml collagen containing calcium ions. Figure 6 e shows 12 mg / ml collagen containing calcium ions, stored in a -30°C freezer.

[0132] Figure 7 Provides freshly cross-linked 12mg / ml collagen bio-ink ( Figure 7 a) and 12 mg / ml collagen bio-ink stored at -30°C after crosslinking ( Figure 7 b).

[0133] Figure 8 Plotting a graph containing 18mM Ca on the y-axis 2+ The viscosity of a 6 mg / ml collagen bio-ink with added riboflavin was plotted against the increased shear rate on the x-axis. The shear trimming behavior shown is essential for extrusion 3D printing.

[0134] Figure 9 This is a graph showing the storage / loss modulus of collagen bio-inks incorporating sodium ascorbate and sodium chloride after UV treatment, as a function of time. Both bio-inks have the same collagen concentration. The graph shows that the intensity of photocrosslinking induced by UV irradiation is halved with the addition of ascorbic acid.

[0135] Figure 10 Provides folded collagen gel ( Figure 10 a) and collagen gel that unfolds in water ( Figure 10 Representative image of b).

[0136] Figure 11 The total transmittance of the collagen gel in the wavelength range of 400-700 nm is shown.

[0137] Figure 12 Representative images of structures formed by stacked lines of collagen bio-ink are provided. Figure 12 The image shows a structure formed from 6 mg / ml collagen bio-ink, and the structure was manipulated with tweezers. Figure 12 b shows a structure formed by stacking threads of 12 mg / ml collagen ink, and the structure is manipulated with tweezers.

[0138] Figure 13 Representative images of HCET cells on top of cross-linked collagen bioink are provided. Figure 13 a) and DAPI staining results of a portion of the collagen gel ( Figure 13 b).

[0139] Figure 14 Representative images of HCSCs on cross-linked collagen bio-inks are provided. Figure 14 a) and the degradation of collagen gel inoculated with HCSC thereon ( Figure 14 b).

[0140] Figure 15 Provides cells encapsulated in cross-linked collagen bio-ink on day 1. Figure 15 a) and cells encapsulated in cross-linked collagen bio-ink on day 5 ( Figure 15 Representative image of b).

[0141] Figure 16 Results of cell delivery experiments using hCSCs are provided. Images show the results 1 day after crosslinking. Figure 16 a) 3 days ( Figure 16 b) and 7 days ( Figure 16 c) Collagen bio-inks and cells.

[0142] Figure 17 A comparison of BrdU readings of HCET cells in a 96-well plate is shown, cells on collagen gel (left) / cells on a tissue culture plate (right) (p = 0.04).

[0143] Figure 18 A comparison of BrdU readings of hCSC cells in a 96-well plate is shown, cells on collagen gel (left) / cells on a tissue culture plate (right) (p = 0.01).

[0144] Figure 19 Representative images of immortalized human corneal endothelial cells on cross-linked, type IV collagen-infused bio-ink at a concentration of 6 mg / ml are provided. The cells reached fusion within 7 days.

[0145] Figure 20 The comparison of BrdU readings of corneal stromal cells in a 96-well plate is shown, with cells on FBS-infused collagen gel (left) / cells on uninfused collagen gel (right) (p = 0.04).

[0146] Figure 21 The invention illustrates filling a pig's cornea with the composition of the present invention. Figure 21 In section a, a 2mm trephine drill is used to form the hole. Figure 21 In step b, after rinsing, the wells were filled with 6 mg / ml collagen bio-ink.

[0147] Figure 22 Images are provided showing the gaps created in pig corneas: (a) gaps created using a 2mm trephine and (b) gaps filled with 12mg / ml collagen ink.

[0148] Figure 23 Images are provided showing the gaps created in pig corneas: (a) the gap created using a 4mm trephine and (b) the gap filled with collagen ink.

[0149] Figure 24 A non-limiting example of an IOP simulation system with a UV curing device is provided.

[0150] Figure 25 Images showing the closure of a pig's cornea are provided. Figure 25 A shows a 6 mg / ml collagen bio-ink for sealing corneal perforations. Figure 25 In b and 25c, the 12 mg / ml collagen bio-ink failed to adhere.

[0151] Figure 26 Images showing the closure of a pig cornea are provided. (a) A 1.5 mm diameter perforation; (b) The closure gap is shown by 12 mg / ml collagen bio-ink.

[0152] Figure 27 Images showing the closure of a pig cornea are provided. (a) A 2 mm diameter perforation; (b) The gap is closed with 12 mg / ml collagen bio-ink.

[0153] Figure 28 A graph showing the volume relationship between part A and part B in crosslinkable collagen ink is provided.

[0154] Figure 29 An image of the generated thin collagen membrane is provided.

[0155] Figure 30 Images of the thicker collagen structure before rinsing are provided.

[0156] Figure 31 Images showing the pores of the 3D-printed mesh structure are provided.

[0157] Figure 32 Calcein-AM staining was provided to show the structure of cells loaded 2 weeks after printing. Invention Details

[0159] The inventors have developed printable collagen bio-inks using unmodified type I collagen, which possess mechanical and structural properties that facilitate their application to tissues in a structured form. In particular, the compositions of this invention can be used to apply collagen gels to tissues (e.g., the eye) using two-dimensional or three-dimensional (extrusion) bioprinting techniques. This composition provides a means of delivering pharmaceutical agents to biological targets (e.g., organs, tissues, cells). While suitable for application to the cornea, the compositions described herein provide a platform for numerous applications in the fields of tissue closure and pharmaceutical delivery by providing, for example, structural support, living cells, and other factors.

[0160] There is a need in the art for effective collagen-derived occlusives and adhesives for surfaces such as the ocular surface and corneal stroma. The compositions described herein are based on natural type I collagen, which, in the case of the cornea, can remodel the major proteins in this tissue. The compositions of the present invention are also ideal agents for the delivery of a variety of growth factors and other pharmaceutical agents. The compositions described herein can utilize biomaterials that mimic in vivo tissues and act as scaffolds for cell proliferation, and / or promote the self-regeneration of cells and their surrounding matrix through manipulation of conditions. In the context of their applicability to ocular tissues, for example, the inventors have overcome the difficulty of creating a matrix that reflects the structural integrity of the tissue being treated (e.g., the cornea) while maintaining transparency and remaining porous and biocompatible enough to allow the infiltration, migration, and / or proliferation of corneal cells and growth factors.

[0161] The challenge at the time of this invention lay in striking a balance between providing the nutritional needs of damaged tissue and meeting the structural, mechanical, and physical requirements of damaged tissue (e.g., ocular tissues such as the cornea). This invention provides improved compositions and methods for delivering pharmaceutical agents to a variety of biological targets. Not limited to any particular application, the compositions can be used for tissue closure and pharmaceutical agent delivery to biological targets.

[0162] Composition for delivery of biological agents

[0163] This invention provides compositions suitable for delivering pharmaceutical agents to biological targets such as tissues and cells. The compositions can also be used as occlusive agents and / or adhesives for said biological targets.

[0164] This composition utilizes a scaffold material to provide structural support when applied to biological targets (e.g., tissues, membranes, cells, organs) to facilitate drug delivery to the biological targets.

[0165] There are no particular restrictions on the one or more specific materials used to create the scaffold.

[0166] For example, the scaffold can be a collagen scaffold. These can be generated, for example, by using type I collagen in the composition. The type I collagen used can be unmodified compared to its naturally occurring counterpart.

[0167] The compositions of the present invention may optionally further comprise ions and / or one or more ion sources. Non-limiting examples of suitable ions include calcium ions and sodium ions. Non-limiting examples of suitable ion sources include compounds containing calcium (e.g., calcium chloride) and compounds containing sodium (e.g., sodium chloride). Calcium ions and sodium ions may be present together or separately in the compositions of the present invention.

[0168] The inventors have determined optimal relative concentrations of type I collagen, sodium ions, and / or calcium ions in the compositions used in this invention, some of which are described in the examples and claims of this application. It should be understood that the disclosed relative concentrations of type I collagen, sodium ions, and / or calcium ions are merely exemplary.

[0169] The composition may contain 1-20 mg / ml of type I collagen, 0.07-0.5 M sodium ions, and / or 0.008-0.4 M calcium ions. In some embodiments, the composition may contain 1-20 mg / ml of type I collagen, 0.07-0.3 M sodium ions, and / or 0.008-0.1 M calcium ions. In some embodiments, the composition may contain 3-15 mg / ml of type I collagen, 0.135-0.3 M sodium ions, and / or 0.008-0.1 M calcium ions. In some embodiments, the composition may contain 3-15 mg / ml of type I collagen, 0.135-0.2 M sodium ions, and / or 0.01-0.05 M calcium ions. In some embodiments, the composition may contain 4-12 mg / ml of type I collagen, 0.135-0.16 M of sodium ions, and / or 0.015-0.03 M of calcium ions. In some embodiments, the composition may contain 5-10 mg / ml of type I collagen, 0.135-0.14 M of sodium ions, and / or 0.018-0.02 M of calcium ions. In some embodiments, the composition may contain 15 mg / ml of type I collagen and more than 0.135 M of sodium ions and / or more than 0.018 M of calcium ions.

[0170] The compositions of the present invention may further comprise one or more crosslinking agents. In some embodiments of the invention, the crosslinking agent is riboflavin. Riboflavin may be present at a concentration of 0.01-0.5% (w / v). Riboflavin may be activated using light, such as UV light or blue light, to crosslink the composition. Those skilled in the art will know of other suitable photocrosslinking agents and light sources, such as rose red dye and green light, both of which have been approved for various applications on the cornea. In some embodiments, rose red is used as a photocrosslinking agent and is activated by green light. In some embodiments, rose red is used as a photocrosslinking agent and is activated by white light. In some embodiments, 0.01-0.5% (w / v) of rose red is used for photocrosslinking. In some embodiments, the composition provided by crosslinking collagen bio-ink with rose red and a suitable light source will be colored. In some embodiments, the color will be pink. These colored compositions may be used to monitor collagen metabolism in tissues or for other uses where tracking collagen activity is required.

[0171] In some embodiments of the present invention, the crosslinking agent is fibrinogen and / or thrombin. The concentration used may be 1.6-6 mg / ml fibrinogen and 1-5 U / mL thrombin. Alternatively, the concentration used may be 0.1-20 mg / ml fibrinogen and 2-20 U / mL thrombin. The inventors have determined the optimal relative concentrations of fibrinogen and thrombin for use in the compositions of the present invention described in the claims of this application. It should be understood that the disclosed relative concentrations of fibrinogen and thrombin are merely exemplary.

[0172] The compositions of the present invention may include platelet lysates. Platelet lysates may, for example, be mammalian platelet lysates (e.g., generated using platelets from humans, dogs, felines, bovines, pigs, horses, sheep, goats, rodents, hares, hamsters (cricetines), or mustelids, or any combination thereof). The source of platelets used to generate the platelet lysates generally depends on the specific purpose for which the composition is intended. As is known to those skilled in the art, platelet lysates are generated by separating platelets, lysing them, and removing cell debris. The composition and applications of platelet lysates have been well analyzed (see, for example, Burnouf et al., Biomaterials. January 2016; 76:371-87).

[0173] In some embodiments, the composition does not contain an anticoagulant or is substantially free of an anticoagulant that may be present only in trace amounts. Non-limiting examples of such anticoagulants include heparin, vitamin K antagonists (e.g., warfarin, coumarin), rivaroxaban, edoxaban, apixaban, dabigatran, etc. In some embodiments, the platelet lysate contains less than: 10% (v / v), 9% (v / v), 8% (v / v), 7% (v / v), 6% (v / v), 5% (v / v), 4% (v / v), 3% (v / v), 2% (v / v), 1% (v / v), or 0.5% (v / v) of anticoagulant.

[0174] The compositions according to the invention may include cells. Cells may be, for example, mammalian cells (e.g., human cells, canine cells, feline cells, bovine cells, porcine cells, horse cells, sheep cells, goat cells, murine cells, rabbit cells, hamster cells, weasel cells, or any combination thereof). The type of cell used generally depends on the specific purpose for which the composition is to be used. For example, the cells may be cells of the same tissue type to which the composition is to be applied (e.g., ocular surface cells, including central and / or peripheral corneal epithelial cells, bulbar and / or palpebral conjunctival epithelial cells, palpebral conjunctival stroma and / or palpebral margin cells; skin cells, including but not limited to keratinocytes, melanocytes, Merkel cells, and Langerhans cells; and neural tissue cells, including but not limited to neurons and glial cells). Other examples include epithelial cells, corneal cells, neuronal cells, and endothelial cells. In some embodiments, the cells may be hematopoietic stem cells, bone marrow stem cells, neural stem cells, epithelial stem cells, skin stem cells, muscle stem cells, adipose stem cells, pluripotent stem cells, induced pluripotent stem cells, embryonic stem cells, mesenchymal stem cells, or any combination thereof. In some embodiments, the cells may be neuronal cells.

[0175] The platelet lysate and / or cells of the composition may be autologous (i.e. derived from the given subject who is to receive the composition) or allogeneic (i.e. donor-derived).

[0176] The compositions of the present invention may contain essential and / or non-essential amino acids. Non-limiting examples of suitable essential amino acids include isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, cysteine, tyrosine, histidine, and arginine.

[0177] The compositions of the present invention may contain additional components (e.g., one or more pharmaceutical agents), including but not limited to fibronectin, anesthetics, antibiotics, hormones (e.g., insulin), growth factors (e.g., human epidermal growth factor (hEGF), platelet-derived growth factor, vascular endothelial growth factor, fibroblast growth factor (FGF), epithelial growth factor, transforming growth factor [including β], and connective tissue growth factor), fibrin stabilizing factors (e.g., factor XIII), one or more matrix proteins (e.g., collagen [such as type IV collagen], laminin, integrin), vitamins (e.g., vitamin C), glycoproteins (e.g., transferrin), fetal bovine serum (FBS), human serum, platelet lysates, human platelet lysates, and any combination thereof. In some embodiments, the composition comprises a culture medium containing ions and amino acids.

[0178] The compositions of the present invention may include other suitable components, including water and / or culture media (e.g., DMEM, DMEM / F-12, MEM, CnT-PR). The culture media may contain any one or more of the following: glycine, L-alanine, L-arginine hydrochloride, L-asparagine monohydrate, L-aspartic acid, L-cysteine ​​hydrochloride monohydrate, L-cysteine ​​dihydrochloride, L-glutamic acid, L-glutamine, L-histidine hydrochloride monohydrate, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine disodium dihydrate, L-valine, vitamins, biotin, choline chloride, D-calcium pantothenate, folic acid, nicotinamide hydrochloride pyridoxine, riboflavin, thiamine hydrochloride, vitamin B12, i-inositol, inorganic salts, calcium chloride (C The composition includes: anhydrous sodium chloride (NaCl2), copper sulfate (CuSO4-5H2O), ferric nitrate (Fe(NO3)3-9H2O), ferric sulfate (FeSO4-7H2O), anhydrous magnesium chloride, anhydrous magnesium sulfate (MgSO4), potassium chloride (KCl), sodium bicarbonate (NaHCO3), sodium chloride (NaCl), anhydrous disodium hydrogen phosphate (Na2HPO4), sodium dihydrogen phosphate (NaH2PO4-), zinc sulfate (ZnSO4-7H2O), other components, D-glucose (dextrose), sodium hypoxanthine, linoleic acid, lipoic acid, putrescine dihydrochloride, sodium pyruvate, thymidine, or any combination thereof. In some embodiments, the ions are provided as components of the ionic salts included in the composition.

[0179] In some embodiments of the invention, the composition is transparent. Alternatively or additionally, the composition may have the ability to retain or substantially retain its shape / structure after printing.

[0180] Non-limiting properties of the composition include one or more of the following:

[0181] - Non-Newtonian shear-thinning fluid properties, thus the viscosity of the composition can decrease with increasing shear rate. In some embodiments, the viscosity of the composition can be in the range of 0.01 to 1000 Pa·s at room temperature.

[0182] - It does not impede or substantially impede the optical sharpness of vision caused by light transmission, for example, more than 90% in the visual color range of 400-700nm.

[0183] - Suitability for 2D and / or 3D printing (e.g., bioprinting / extrusion printing), with the ability to retain or substantially retain the shape / structure after printing.

[0184] - Suitable for printing, while maintaining the vitality of cells within the composition during the printing process.

[0185] - Capabilities provided in two-dimensional or three-dimensional structures, with or without living cells.

[0186] - The ability to maintain and / or promote cell growth (e.g., to maintain and / or promote the expansion of primary human cells such as epithelial cells, corneal cells, neurons, and endothelial cells).

[0187] - The ability to promote the formation of globular organoids.

[0188] - The ability of cells to degrade over time (e.g., 2-7 days).

[0189] - Maintain cell viability over time (e.g., 7 days at 34°C).

[0190] - The ability to adhere to various surfaces, including tissues, organs, and membranes (e.g., mammalian and human tissues, organs, and membranes).

[0191] Preparation of the composition

[0192] Typically, the compositions of the present invention can be prepared by combining a variety of different formulations. Lyophilized type I bovine collagen can be used to prepare the compositions. Alternatively or alternatively, human collagen can be used. Type I collagen can be neutralized before the addition of ions and other components of the composition. In some embodiments of the invention, one or more cross-linking agents are combined with a base to form part A, and type I collagen is combined with ions such as sodium ions and / or calcium ions to form part B; parts A and B are mixed to form a solution, which is then applied to a surface. In some embodiments, parts A and B are mixed in a ratio of (1-20):(200-300). In a further embodiment, the ratio is 9:250. Those skilled in the art will recognize that various methods can be used to prepare the compositions of the present invention and various buffer solutions can be used to maintain the collagen at physiological pH and preserve its solubility.

[0193] In some embodiments of the invention, the cross-linking agents are fibrinogen and thrombin. In some embodiments, the type I collagen formulation containing thrombin can be stored separately from the type I collagen formulation containing fibrinogen, and the two formulations can be combined before or during the application of the composition.

[0194] In some embodiments, type I collagen plus fibrinogen and type I collagen plus thrombin formulations can be provided by establishing separate flow streams of the two separated components. These streams can be kept continuously flowing for a suitable period of time and oriented to mix with each other at a given point, thereby providing a further flow of mixed components deposited on the biological target. Alternatively, the streams can be oriented to mix with each other at or on the surface of the biological target on which the composition is applied. The compositions of the present invention, using fibrinogen and thrombin as crosslinking agents without the addition of platelet lysate, may be opaque. Such opaque collagen bioinks can be used for a wider range of applications beyond ophthalmology.

[0195] The compositions of the present invention may contain platelet lysates (e.g., mammalian platelet lysates, human platelet lysates). Platelet lysates can be prepared by any suitable method (e.g., by freeze / thaw lysis; see, for example, Chou and Burnouf, ISBT Science Series, Vol. 12, No. 1, February 2017, pp. 168-175). Adding platelet lysates to the compositions of the present invention crosslinked with fibrinogen and thrombin may make the compositions transparent.

[0196] Furthermore, the inventors have observed that the use of anticoagulants during the preparation of platelet lysates (e.g., during culture) can affect the ability of platelet lysates to form the compositions according to the invention. Therefore, in some embodiments of the invention, the platelet lysates used are prepared without an anticoagulant (e.g., heparin) at some or all stages of the preparation method.

[0197] In some embodiments, the present invention provides devices and / or kits that facilitate the separation of different formulations required to form the compositions of the present invention up to their use. In some embodiments, the devices and kits may comprise at least two physically separated compartments, the first compartment containing a collagen solution formulation having ions and thrombin, and the second compartment containing a collagen solution formulation having ions and fibrinogen. In some embodiments, any one or both compartments may contain additional components for generating the composition (e.g., platelet lysate, ions, amino acids, cells, antibiotics, growth factors, vitamins, fibrin stabilizing factors, anesthetics, etc.).

[0198] The apparatus and kit may further include components that provide a method for facilitating the mixing of the two compartments, for example by removing one or more barriers separating the first and second compartments, and / or by piercing the closure or wall of any one or both compartments. Those skilled in the art will readily understand that various arrangements can be made for this purpose.

[0199] Alternatively or concurrently, the device and kit may be configured in a manner that ensures the mixing of two separate formulations during or after the release of the formulation from the device or kit.

[0200] In some embodiments, the device and kit may include an additional compartment containing additional components for generating the composition (e.g., platelet lysate, ions, amino acids, cells, antibiotics, growth factors, fibrin stabilizing factors, anesthetics, etc.). The device or kit may be configured to facilitate the mixing of these additional components with each other and / or with one or more formulations of fibrinogen and / or thrombin during use of the device or kit.

[0201] The device and kit can facilitate the mixing of separated components before, during, or immediately after the components are discharged from the device or kit.

[0202] In some embodiments, the composition is a bio-ink and the device is a three-dimensional (3D) printer (e.g., an extrusion printer).

[0203] In some embodiments of the invention, the crosslinking agent is riboflavin. In some embodiments, riboflavin can be activated by UV light or blue light. A solution that may contain type I collagen and sodium and / or calcium ions can be extruded into a thread, and UV light or blue light can be applied. Additional threads can be applied on top of the first thread to form a structure that will be crosslinked by the crosslinking agent and the application of light.

[0204] In some implementations, photocrosslinking occurs within 15 minutes, 14 minutes, 13 minutes, 12 minutes, 11 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, or 1 minute. The light source used can be 3mW / cm². 2 365nm UV, 10mW / cm 2 Blue light or UV lamp for tissue culture.

[0205] In some embodiments, the photocrosslinking agent is rose red. In some embodiments, rose red is activated by green light. In some embodiments, rose red is activated by white light. In some embodiments, photocrosslinking occurs within 15 minutes, 14 minutes, 13 minutes, 12 minutes, 11 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, or 1 minute. The light source used can be 100 mW / cm². 2 White light. Those skilled in the art will recognize that the light source and parameters can be varied depending on the specific application.

[0206] The collagen gel of the present invention can have different thicknesses depending on the application; for example, the thickness of the gel can be from 50 μm to 5 mm.

[0207] Application of the composition

[0208] The inventors have developed a composition for delivering one or more agents to target tissues and cells, which possesses properties that make it highly suitable for bioprinting. The composition of this invention can be used in applications requiring the delivery of reagents (e.g., natural growth factors, drugs, nanoparticles, and / or cells) and / or the immobilization of individual biological surfaces, and / or in tissue culture methods.

[0209] In some embodiments, the compositions can be used as tissue blocking agents and / or as fixatives for biological structures. They can provide structural and / or nutritional support to tissues. Additionally or alternatively, the compositions can promote the growth of one or more target cell types, including those that can be provided as components of the composition and / or cells present in the target tissue.

[0210] In some embodiments, the composition is colored by crosslinking collagen bio-ink with rose red and a suitable light source. In some embodiments, the collagen composition may be pink. These colored compositions can be used to monitor collagen metabolism in tissues or for other uses where tracking collagen activity is required.

[0211] Although there are no restrictions on the types of tissues to which the composition can be applied, the inventors have demonstrated that the composition is effective in closing ocular tissues.

[0212] For example, this article demonstrates the effectiveness of the composition in sealing corneal tissue. In these embodiments, the composition can be used to promote the proliferation and / or migration of corneal epithelial cells. The composition can, for example, support the multidirectional growth and / or stratification of corneal epithelial cells, which, once a cell monolayer is formed, can be partially or completely biodegraded.

[0213] This invention therefore provides methods for sealing tissues and delivering various agents to biological targets. For example, the target may be located in or around ocular tissues, including the central and / or peripheral corneal epithelium, bulbar and / or palpebral conjunctival epithelium, palpebral conjunctival stroma, and / or eyelid margin tissues.

[0214] Those skilled in the art will understand that various changes and / or modifications can be made to the invention as illustrated in the specific embodiments without departing from the spirit or scope of the invention as broadly described. Therefore, these embodiments are to be considered illustrative rather than restrictive in all respects. Example

[0215] The invention will now be described with reference to specific embodiments, which should not be construed as limiting in any way.

[0216] Example 1: Preparation and Characterization of Collagen Bio-Ink

[0217] Materials and methods

[0218] 1.1 Preparation of Type I Collagen Solution

[0219] Type I collagen powder (bovine skin, Sigma-Aldrich) was prepared according to the manufacturer's instructions. The powder was dissolved in 0.1M acetic acid solution (pH = 2.72) with continuous stirring at 800 rpm for 16 hours. The mixture was then visually inspected to confirm complete dissolution of the collagen powder. The collagen solution was then transferred to 1.5 ml Eppendorf tubes and stored in a refrigerator.

[0220] 1.2 Effect of Ions on the Stability of Neutral Type I Collagen Solutions

[0221] For each solution generated, 90 parts of collagen solution were neutralized with 2.7 parts of 5M NaOH, and then mixed with 7.3 parts of various salt solutions. The solutions tested included 1× phosphate-buffered saline (PBS, composed of NaCl, KCl, Na₂HPO₄, and KH₂PO₄), NaCl, KCl, Na₂HPO₄, KH₂PO₄, and sodium ascorbate (NaC₆H₇NO₆). The concentrations of the ions for each test are listed in Table 1. Solutions were left on the workbench for 1 hour, 1 day, and 1 week before examining for precipitation formation.

[0222] Table 1: Concentration of ions used in the test

[0223]

[0224] 1.3 Generation of photocrosslinkable collagen bio-ink

[0225] - Dissolve riboflavin in a 20 mg / ml CaCl2 solution.

[0226] 0.1% (w / v) and 0.2% (w / v) riboflavin solutions were prepared by adding 0.5 and 1 mg of riboflavin powder, respectively, to 500 μL of 2 mg / mL CaCl2 solution. The tubes were vortexed for 2 minutes, and the precipitation was examined by centrifuging the tubes in a miniature centrifuge for 1 minute.

[0227] Riboflavin powder (Sigma-Aldrich) was added to a freshly prepared ion-added neutral collagen solution at a final concentration of 1 mg / ml. Vortexing was necessary to completely dissolve the riboflavin powder in the collagen bio-ink. The riboflavin-added collagen bio-ink was then frozen at -30°C in foil-wrapped Eppendorf tubes.

[0228] The collagen bio-ink with added riboflavin underwent photocrosslinking via various light sources, including 3mw / cm². 2 365nmUV, 10mW / cm 2 Blue light and tissue culture hood UV lamp.

[0229] 1.4 Generation of photocrosslinkable collagen-based bio-ink

[0230] Type I collagen powder (human skin, Sigma-Aldrich) was dissolved in 0.1M acetic acid solution by vortexing for 10 minutes. The mixture was then visually inspected to confirm that the collagen powder was completely dissolved. The collagen solution was then transferred to 0.5ml Eppendorf tubes for further processing.

[0231] The human collagen solution (90 parts) was neutralized with 2.7 parts of 5M NaOH, and then mixed with 7.3 parts of various salt solutions. Riboflavin powder (Sigma-Aldrich) was then added to a final concentration of 1 mg / ml. The solution was then filtered through a 3mw / cm... 2 A 365nm UV light was used to photocrosslink a human collagen solution containing riboflavin for 2 minutes. Rose red powder (Sigma-Aldrich) was added to the freshly prepared neutral collagen solution (the same as used for riboflavin) at a final concentration of 1 mg / ml. The collagen ink containing rose red emitted 100 mw / cm². 2 The white LED light is further cross-linked for 2 minutes.

[0232] 1.5 Using rose red and green light to generate photocrosslinkable collagen-based collagen bio-ink

[0233] Rose cinnabar powder (Sigma-Aldrich) was added to the freshly prepared neutral collagen solution at a final concentration of 1 mg / ml. The collagen ink containing rose cinnabar emitted 100 mw / cm². 2 Crosslinking of white light LEDs for 2 minutes.

[0234] result

[0235] 1.6 Preparation of Type I Collagen Solution

[0236] Up to 15 mg of type I collagen powder (bovine skin, Sigma-Aldrich) was found to be completely soluble in 1 ml of 0.1 M acetic acid without precipitation.

[0237] 1.7 Effect of Ions on the Stability of Neutral Type I Collagen Solutions

[0238] The results showed that the minimum Na content for a stable neutral collagen solution was... + The concentration is 136.9 mM, excluding the Na in the 135 mM NaOH. + Ca 2+ The lowest concentration is 18 mM ( Figure 1 ). with Na at or above the minimum concentration. + The combined ion results also showed stable solubility of type I collagen. The conclusion is that including NaCl (at a minimum concentration of 68.4 mM) or CaCl2 (at a minimum concentration of 18 mM) is optimal for composing soluble collagen bio-inks.

[0239] 1.8 Generation of photocrosslinkable collagen bio-ink

[0240] - Dissolve riboflavin in a 20 mg / ml CaCl2 solution.

[0241] Riboflavin powder is readily soluble in 2 mg / ml CaCl2 (0.1% w / v, 0.5 mg in 500 μL solution) to form a clear yellow solution. Figure 2 a) However, 1.0 mg of riboflavin did not completely dissolve (0.2% w / v), producing an opaque solution. Figure 2 b).

[0242] like Figure 3 As shown, the collagen bio-ink prepared according to the methods described in Sections 1.1 to 1.3 can pass through a flow rate of 3 mw / cm³ within 2 minutes. 2 365nm UV, 10mw / cm 2 470nm blue light crosslinks the collagen gel, forming it that adheres to the glass slide. The tissue culture hood's sterilizing UV (254nm UV) crosslinks the collagen bio-ink within 15 minutes.

[0243] 1.9 Generation of photocrosslinkable collagen-based bio-ink

[0244] The human collagen-based bioink prepared according to Section 1.4 can pass through a flow rate of 3 mw / cm in 2 minutes. 2 365nm UV crosslinking forms a collagen gel that adheres to the glass slide. Figure 4 ).

[0245] 1.10 Using rose red and green light to generate photocrosslinkable collagen-based collagen bio-ink

[0246] Type I collagen solutions with added rose red as a photocrosslinking agent can pass through 100 mw / cm in 2 minutes. 2 White light crosslinks to form a pinkish collagen gel that adheres to the glass slide. Figure 5 ).

[0247] Unlike riboflavin crosslinking, the color does not fade after rinsing because the rose red molecules bind to type I collagen. Collagen bioinks crosslinked in this way remain viscous and can be used to monitor collagen metabolism in tissues or for other applications requiring tracking of collagen activity.

[0248] The above non-limiting examples demonstrate the use of up to 15 mg / ml of natural type I collagen in the preparation of transparent collagen bio-inks, which can crosslink to form a transparent collagen gel in just two minutes. This example also demonstrates the use of human collagen in the preparation of bio-inks.

[0249] The above embodiments also demonstrate that using other ions instead of calcium is unlikely to yield good results. Sodium must be present for neutralization purposes. The addition of calcium increases the storage modulus of the ink. Other ions (such as potassium) must be added in amounts far exceeding physiological ranges to stabilize the collagen solution, and are therefore unsuitable for the collagen bio-ink of this invention.

[0250] Example 2: Mechanical Testing of Collagen Bio-Ink

[0251] Materials and methods

[0252] 2.1 Photorheology of Collagen Bio-ink

[0253] The rheological properties of freshly prepared collagen bioinks and frozen collagen bioinks were examined using photorheological testing. For the freshly prepared collagen bioinks, tests were conducted with PBS and CaCl2 at different collagen concentrations (Table 2, samples 1-4). Riboflavin powder (Sigma-Aldrich) was mixed with collagen solution according to the method described in Section 1.3. The crosslinking state (expressed as storage modulus) was evaluated using a TA AR-G2 rheometer coupled with an Omnicure 1000 UV lamp with a fixed wavelength of 365 nm. The crosslinking was performed using 12 mg / ml collagen solution and 18 mM CaCl2. 2+ Collagen bio-ink prepared with 1 mg / mL riboflavin was stored at -30°C for 1 week and then thawed before photorefractive testing (Table 2, Sample 5).

[0254] Table 2: Collagen bio-ink samples used for photorheological testing

[0255] Sample number Type I collagen concentration buffer solution 1 12mg / ml 1×PBS 2 12mg / ml <![CDATA[18mM CaCl2]]> 3 6mg / ml <![CDATA[18mM CaCl2]]> 4 3mg / ml <![CDATA[18mM CaCl2]]> 5 12mg / ml <![CDATA[18 mM CaCl2 (frozen)]]>

[0256] A 20mm flat plate geometry (geometric gap = 300μm) was used. Collagen bio-ink (120μL) was loaded onto the plate. Testing was conducted at room temperature. The UV intensity was adjusted to 3.2mw / cm. 2 The collagen bio-ink was agitated at 0.2 Hz and 1% strain for 10 minutes at 34°C. UV was applied to the collagen bio-ink at the 2-minute mark.

[0257] Record the changes in storage modulus and loss modulus of collagen bio-ink over time. Observe the cross-linked collagen bio-ink with the naked eye.

[0258] 2.2 Rotation Test of Collagen Bio-Ink

[0259] For the rotation test, 60 μL containing 18 mM Ca 2+ The 6 mg / ml collagen bio-ink underwent shear force at 34 °C with shear rates increasing from 0.1% to 20% from its geometry. The viscosity of the collagen bio-ink was monitored.

[0260] 2.3 Measurement of storage modulus of collagen bio-ink incorporating ascorbate

[0261] Storage modulus was measured using a TA AR-G2 rheometer with a 15 mm diameter corn-and-plate geometry (geometric gap = 55 μm) for collagen bio-ink incorporating ascorbate (6 mg / ml collagen bio-ink containing 68.4 mM sodium ascorbate) and a control (6 mg / ml collagen bio-ink containing 68.4 mM NaCl). Prior to storage modulus measurement, a 3 mW / cm² rheometer was used. 2The collagen bio-ink was treated with 365nm UV for 16 seconds. For storage modulus measurement, 60 μL of collagen bio-ink was oscillated at 0.2 Hz and 1% strain for 10 minutes. The storage modulus and loss modulus of the collagen bio-ink were recorded.

[0262] 2.4 Preparation of thin films via cross-linked collagen bio-ink

[0263] A small amount of collagen bio-ink with added riboflavin was transferred onto the surface of a glass slide. The collagen bio-ink on the slide was further spread to cover as much area as possible without leaving gaps in the diffusion area. This was done by carefully applying the bio-ink using a pipette tip. Then, it was passed through a 3mw / cm... 2 The collagen bio-ink was crosslinked for 2 minutes under a 365nm UV curing lamp, followed by multiple washes in PBS to remove riboflavin. The thickness of the crosslinked gel was measured. The flexibility and strength of the crosslinked gel were observed by lifting it with tweezers and immersing it in Milli-Q water.

[0264] 2.5 Spectrophotometric Measurement of Crosslinked Collagen Bioink

[0265] The optical properties of films produced by crosslinking collagen bio-ink using the method described in Section 1.3, wherein the collagen bio-ink is composed of 18 mM Ca 2+ The sample consisted of a 6 mg / ml collagen solution containing 0.1% riboflavin. The spectrophotometer was set to measure total transmittance in the visible light range, and a circular 3D-printed sample holder was used to read the background. The collagen membrane was carefully transferred onto the sample holder, which was then inserted into the spectrophotometer. The total transmittance curve was obtained.

[0266] 2.6 Use line stacking test to check initial printing potential

[0267] Set up a printing stage on the workbench, with the printing area being the center of a 100mm culture dish with a diameter of 2cm. Use 3mw / cm 2 UV treatment was continuously applied to the printing area. A collagen solution containing 6 mg / ml (containing 18 mM Ca) was slowly extruded via a 20 μL pipette. 2+ Collagen bio-ink containing 0.1% riboflavin was used to draw 10mm × 1mm lines. After a further UV treatment of 1 minute, another line was drawn on top of the previous line as described earlier. A total of 10 stacks were performed to form a 3-D structure. A simple 3-D structure was created by stacking the 10 drawn lines one by one. The shape and integrity of the structure were checked by manipulating it with tweezers.

[0268] result

[0269] 2.7 Photorheological properties of collagen bio-ink

[0270] like Figure 6 As shown in Figure a, the storage modulus of 12 mg / ml collagen bio-ink in 1×PBS increased from 1.7 Pa to 10 Pa after 1 minute of UV irradiation, demonstrating photocrosslinking. The resulting gel is a relatively weak gel with a storage modulus of 10 Pa. Figure 6 a). For 18mM Ca 2+ Collagen solutions of the same concentration in the medium began to crosslink rapidly after 1.5 minutes of UV irradiation, resulting in a stronger gel with a storage modulus of 70 Pa. Figure 6 b).

[0271] For containing 18mM Ca 2+ The 6 mg / ml collagen bio-ink, after cross-linking, has a storage modulus of 40 Pa, while containing 18 mM Ca... 2+ The storage modulus of the 3 mg / ml collagen bio-ink was 1.5 Pa. There was no difference in the minimum UV irradiation time required for rapid crosslinking. Figure 6 c and Figure 6 d).

[0272] Collagen samples (6 mg / ml, fortified with riboflavin, containing 18 mM Ca) stored in a -30°C freezer and thawed before testing 2+ The collagen bio-ink can undergo photocrosslinking within 1.5 minutes. Compared to a freshly prepared sample with the same composition, the crosslinked collagen ink exhibits a 5-fold increase in storage modulus. Figure 6 d and Figure 6 e). Based on visual observation, it is similar to a freshly prepared sample with the same composition. Figure 7 a) In comparison, collagen samples stored in a -30°C freezer formed a more robust gel after cross-linking. Figure 7 b).

[0273] 2.8 Rotation Test of Collagen Bio-Ink

[0274] The results of the rotation test showed that 6 mg / ml, with added riboflavin and containing 18 mM Ca 2+ The collagen bio-ink exhibits shear-thinning behavior, with its viscosity decreasing with shear rate. Figure 8 This property is necessary for extrusion 3D printing.

[0275] 2.9 Measurement of storage modulus of collagen bio-ink incorporating ascorbate

[0276] Compared to collagen bio-inks with the same collagen concentration but without ascorbate (storage modulus of 300 kPa), collagen bio-inks incorporating ascorbate showed almost half the storage modulus (120 kPa) after 10 minutes of UV treatment. Figure 9 The intensity of photocrosslinking induced by UV irradiation is halved after the addition of ascorbic acid.

[0277] 2.10 Preparation of collagen gels of different thicknesses

[0278] The minimum thickness of the generated collagen gel was measured at 100 μm. As observed with the naked eye, the generated collagen gel is transparent. It can be easily picked up with tweezers without breaking in a folded manner. Figure 10 a). Without additional force, the gel unfolds upon immersion in Milli-Q water. Figure 10 b).

[0279] 2.11 Spectrophotometric Measurement of Crosslinked Collagen Bioink

[0280] Spectrophotometric results showed that the decrease in light intensity passing through the cross-linked collagen bio-ink was less than 10%. Figure 11 The spectrum shows that the total transmittance of the collagen gel in the visible light range exceeds 90%. Figure 11 ).

[0281] 2.12 Use line stacking test to check initial printing potential

[0282] Using the method described in Section 2.6, 10 6 mg / ml collagen bio-ink lines can be stacked to form a structure, and can be picked up with tweezers without separating the layers. Figure 12 a). Collagen bio-ink with a collagen concentration of 12 mg / ml can also be stacked into 10 layers to form a similar structure, and this structure can also be clamped without structural damage. Figure 12 b).

[0283] The results of the above non-limiting embodiments demonstrate that the collagen bioink of the present invention is capable of gelation because its storage modulus is greater than its loss modulus. The bioink also exhibits shear thinning, a desirable property for extrusion-based 3D bioprinting.

[0284] The reduction in light brightness passing through the cross-linked collagen bio-ink is less than 10%, thus demonstrating that it does not impede or substantially impede optical sharpness of vision caused by light transmission. This example demonstrates that the collagen gel has a total transmittance of over 90% in the visual color range of 400-700 nm.

[0285] Example 3: In vitro biocompatibility test of collagen bio-ink

[0286] Materials and methods

[0287] 3.1 Culture of human corneal epithelial cell line (HCET) and human corneal stromal cell line (HCSC) using collagen gel

[0288] Transformed human corneal epithelial cells were cultured in HCET growth medium at 37°C and 5% CO2. This medium consisted of DMEM / F12 (Thermo Fisher Scientific), 5% (v / v) fetal bovine serum (FBS) (Sigma-Aldrich), and 10 ng / ml human epidermal growth factor (hEGF) (Thermo Fisher Scientific). Cells were passaged for experiments after reaching 85%-90% confluence.

[0289] Human corneal stromal cells were obtained by culturing donor tissue explants in 10% FBS cell culture medium, which was 10% FBS in DMEM / F12. Cells were passaged for experiments after reaching 80%-85% confluence.

[0290] In this experiment, the collagen bio-ink consisted of 6 mg / ml type I collagen and 1% riboflavin, and was prepared using 18 mM CaCl2. 2 ml of the collagen bio-ink was transferred to a 35 mm culture dish, UV cross-linked under a tissue culture hood for 1 hour, and then rinsed multiple times with PBS to remove the pale yellow color caused by riboflavin.

[0291] HCET cells were used at a rate of 5 × 10 4 cells / cm 2 The inoculation density was applied to the top of the collagen gel. The culture dish was stored in a 37°C, 5% CO2 incubator. HCSCs were also inoculated at a density of 5 × 10⁻⁶. 4 cells / cm 2 Cells were seeded onto collagen gel at the specified seeding density and stored in a 37°C, 5% CO2 incubator. The cell culture medium was changed every two days. Cell growth was observed using an Olympus IX71 inverted microscope.

[0292] Once the cells had fused on the collagen gel surface, a portion of the collagen gel was removed using a 4 mm diameter trephine. The harvested collagen gel slides were fixed and frozen into 20 μm sections. The cell nuclei in the sections were stained with DAPI.

[0293] 3.2 Cell delivery experiments using HCET

[0294] A collagen bioink composed of 6 mg / ml type I collagen, 1% riboflavin, and 18 mM CaCl2 was used in this experiment. The collagen bioink was mixed with HCET cells in its growth medium to obtain 1 × 10⁻⁶ cells. 6HCET cells / ml were used to draw three lines in a 35mm culture dish by slowly transferring the cell-containing collagen bio-ink using a 200μL pipette tip. The three collagen bio-lines were then treated with 3mw / cm for 2 minutes. 2 365nm UV treatment. After UV treatment, add 2ml of cell culture medium to the culture dish. Store the culture dish in a 37°C incubator with 5% CO2. Change the cell culture medium every two days. Observe the culture dish daily using an Olympus IX71 inverted microscope.

[0295] 3.3 Cell delivery experiments using hCSC

[0296] A collagen bioink composed of 6 mg / ml type I collagen, 1% riboflavin, and 18 mM CaCl2 was used in this experiment. The collagen bioink was mixed with human corneal stromal cells (hCSCs) in its growth medium to obtain 5 × 10⁵ cells. 4 A density of cells / ml was established. Then, using a 200μL pipette with a 200μL tip, collagen bio-ink containing cells was slowly transferred to draw a line in a 35mm culture dish. The collagen bio-ink line was then subjected to 3mw / cm for 1 minute. 2 365nm UV treatment. After UV treatment, add 2ml of cell culture medium to the culture dish. Store the culture dish in a 37°C incubator with 5% CO2. Change the cell culture medium every two days. Observe the culture dish daily using an Olympus IX71 inverted microscope.

[0297] 3.4 Cell proliferation assay using bromodeoxyuridine / 5-bromo-2'-deoxyuridine

[0298] 5-Bromo-2'-deoxyuridine (BrdU) is a chemical that can be incorporated into DNA during its synthesis, and therefore can be used to measure cell proliferation. A higher BrdU reading indicates a greater amount of DNA synthesized, which means more cell proliferation.

[0299] The BrdU Cell Proliferation ELISA Kit (chemiluminescence) from Abcam was used. HCET cells (1000 cells / well) or human corneal stromal cells (hCSC) (3000 cells / well) were seeded into 96-well plates according to the manufacturer's instructions. Cells were tested under two conditions: cells seeded on top of collagen gel with BrdU staining (Experimental Group 1), cells seeded on top of collagen gel without BrdU staining (BrdU Background 1), cells seeded without collagen gel but with BrdU staining (Experimental Group 2), and cells seeded without collagen gel and BrdU staining (BrdU Background 2) (Table 3).

[0300] Table 3: Experimental groups of the BrdU cell proliferation ELISA kit for HCET and human corneal stromal cells

[0301]

[0302] In this experiment, the collagen bio-ink used to form collagen gel consisted of 6 mg / ml collagen, 1% riboflavin, and 18 mM Ca. 2+ composition.

[0303] HCET cells were cultured in DMEM / F12 containing 5% FBS. BrdU was added to the designated wells 2 hours before the end of culture. Human corneal stromal cells were cultured in serum-free corneal cell growth medium consisting of Dürbeco Modified Eagle Medium / Nutrient Mixture F-12 (DMEM / F12) (Thermo Fisher Scientific), 1% (v / v) insulin-transferrin-selenoethanolamine (ITS-X) (Thermo Fisher Scientific), 10 ng / ml basic fibroblast growth factor (FGF-basic) (Thermo Fisher Scientific), and 1 mM ascorbic acid. BrdU was added to the designated wells 4 hours before the end of culture.

[0304] result

[0305] 3.5 Culture of human corneal epithelial cell line (HCET) and human corneal stromal cell line (HCSC) using collagen gel.

[0306] Phase-contrast microscopy showed that HCET cells could achieve fusion on the surface of collagen gel within 7 days of cell culture. Figure 13 a). DAPI staining of cross-sections of collagen gel inoculated with cells (with staining of cell nuclei) indicated that some cells migrated into the collagen gel during cell culture. Figure 13 b).

[0307] HCSC also achieved fusion within 7 days. Figure 14 a). When cells reach fusion, degradation of the collagen gel can be observed ( Figure 14 b).

[0308] 3.6 Cell delivery experiments using HCET

[0309] Figure 15 Figure a shows cells encapsulated in cross-linked collagen on day 1. Cell migration and proliferation were observed on day 5, while the structure of the cross-linked collagen bioink remained intact after cell migration. Figure 15 b).

[0310] 3.7 Cell delivery experiments using hCSC

[0311] Figure 16 The images show cell migration and proliferation observed within the collagen gel on day 3, and cells beginning to migrate out of the collagen gel on day 7. No degradation of the collagen gel was observed during this process.

[0312] 3.8 Cell proliferation assay using bromodeoxyuridine / 5-bromo-2'-deoxyuridine

[0313] After incubation with BrdU for 2 hours, the BrdU reading of HCET cells on top of the collagen gel was significantly higher (p = 0.04) than that of cells on the tissue culture plate. Figure 17 After incubation with BrdU for 4 hours, human corneal stromal cells on top of collagen gel also showed significantly higher BrdU readings than cells on tissue culture plates (p = 0.01). Figure 18 ).

[0314] The results of this embodiment demonstrate that the collagen bioink of the present invention exhibits high cell compatibility. When seeded on top of collagen gel, the bioink supports cell proliferation and migration of human corneal epithelial cells and human corneal stromal cells. The results also show that UV photocrosslinking does not affect the survival of the encapsulated cells. These results indicate that the collagen bioink of the present invention can be used for cell delivery.

[0315] Example 4: Generating different versions of collagen bio-ink

[0316] Materials and methods

[0317] 4.1 Preparation of Collagen Bio-ink Incorporated with Type IV Collagen

[0318] Type IV collagen powder was dissolved in a 6 mg / ml acidic collagen solution to a concentration of 0.2 mg / ml. The collagen solution incorporating type IV collagen was further neutralized before the addition of 18 mM calcium ions and 0.1% riboflavin to produce a photocrosslinkable type IV collagen-incorporated collagen ink.

[0319] 4.1.1 Corneal endothelial cell lines cultured on collagen gel surface

[0320] Human corneal endothelial cell line (HCEC-B4G12) was cultured on top of cross-linked collagen bioink incorporating type IV collagen at a cell density of 5 x 10⁻⁶ cells / year. 4 cells / cm 2The culture medium consisted of 5% fetal bovine serum (FCS), 20 μg / ml ascorbic acid, 20 μg / ml insulin, and 10 ng / ml FGF-alkaline in a 1:1 mixture of Ham's F12 nutrient mixture and medium 199 (F99 medium). Cells were cultured in a 37°C, 5% CO2 incubator, with the culture medium changed every two days.

[0321] 4.2 Preparation of FBS-infused collagen bio-ink

[0322] A sterile collagen bio-ink composed of 12 mg / ml type I collagen and 18 mM CaCl2 was used. Nine parts of the collagen ink were mixed with one part FBS to obtain a collagen ink containing 10% FBS. The collagen ink containing 10% FBS was then photocrosslinked to form a collagen gel.

[0323] 4.2.1 Cell proliferation assay

[0324] BrdU cell proliferation assays were performed as described in Section 3.4. Human corneal fibroblasts were cultured in DMEM / F12 medium at a seeding density of 2000 cells / well. BrdU was added to the designated wells 20 hours before the end of the culture period.

[0325] result

[0326] 4.3 Collagen bio-ink incorporating type IV collagen

[0327] The human corneal endothelial cell line (HCEC-B4G12) cultured on top of a cross-linked collagen ink incorporating type IV collagen reached fusion within 7 days. Figure 19 ).

[0328] 4.4 Collagen Bio-ink Incorporated with FBS

[0329] Human corneal stromal cells cultured on the surface of FBS-infused collagen gel showed higher BrdU readings than HCSCs seeded on uninfused collagen gel. Figure 20 This indicates higher cell proliferation.

[0330] The results in this embodiment demonstrate that photocrosslinked collagen gels can be readily prepared by mixing type I collagen bio-ink with type IV collagen. Since type IV collagen is a major component of the Descemet's membrane, to which corneal endothelial cells adhere, this combination could provide further benefits for corneal endothelial cell culture.

[0331] Example 5: Ex vivo filling / sealing of porcine cornea

[0332] Materials and methods

[0333] 5.1 Filling the corneal stroma with collagen bio-ink

[0334] Two types of holes were created in the cornea—stromal pores and perforations. Pig corneas were implanted into the anterior chamber, and a trephine with a diameter of 2 or 4 mm was used to remove up to 0.5 mm of the anterior cornea, resulting in non-penetrating damage to the stromal. At 3 mw / cm 2 365nm UV or 10mw / cm 2 Extrusion under blue light curing yielded collagen with 6 mg / ml and 12 mg / ml concentrations, 1% riboflavin, and 18 mM Ca, as prepared in Section 6.1. 2+ Collagen ink was applied to fill the pores. After the pores were filled, an additional 2 minutes of UV irradiation was applied. The adhesion of the collagen ink was checked by rinsing the cornea under running water for 30 seconds and then drying it with kimwipes.

[0335] 5.2 Seal perforations with collagen bio-ink

[0336] An IOP simulation system was established to measure the burst pressure of a collagen-ink-sealed cornea. The IOP simulation system consisted of a height-adjustable syringe connected to the anterior chamber via a medical polyethylene tube. Pressure was generated by adding water to the syringe and quantified by converting the water level to the mercury level (mmHg). Two pressure points, 22 mmHg and 50 mmHg, were tested for normal IOP and extremely high IOP.

[0337] A perforation was created using a 0.8 mm diameter needle, a 1.5 mm diameter needle, or a 2 mm diameter trephine to penetrate the porcine cornea fixed to the anterior chamber. Water was continuously added to the syringe to maintain pressure. Then, as described in Section 5.1 above, 6 mg / ml and 12 mg / ml collagen bio-ink were applied under UV irradiation or blue light curing to seal the perforation. Milli-Q water was then pumped into the anterior chamber to restore pressure. The cross-linked collagen bio-ink and any leakage were visually inspected.

[0338] result

[0339] 5.3 Filling the corneal stroma with collagen bio-ink

[0340] Non-perforating holes formed in the pig cornea are shown in... Figure 21 a (circle). The stage of the gap and the application of 6 mg / ml cross-linked collagen bio-ink to the pores after rinsing with running water for 30 seconds are shown. Figure 21 b (circle). 12 mg / ml collagen ink produced the same result ( Figure 22 a and Figure 22 b). Collagen bio-ink can also fill larger pores (4mm in diameter) under blue light curing. Figure 23 ).

[0341] 5.4 Seal perforations with collagen bio-ink

[0342] The IOP simulation system used in this example is shown below. Figure 24 Collagen bio-ink with a collagen concentration of 6 mg / ml can seal perforations with a diameter of 0.8 mm and prevent perforation leakage at IOPs of 22 mmHg and 50 mmHg. Figure 25 a). Collagen bio-ink with a collagen concentration of 12 mg / ml failed to adhere to the perforation area at 22 mmHg IOP. Figure 25 b).

[0343] 12 mg / ml collagen ink can seal perforations with a diameter of 1.5 mm and prevent leakage up to 50 mmHg IOP, while 6 mg / ml collagen ink cannot seal perforations. Figure 26 a, Figure 26 b).

[0344] 12 mg / ml collagen ink can seal 2 mm diameter perforations at 22 mm Hg, but cannot prevent leakage at higher IOPs. Figure 27 a, Figure 27 b).

[0345] The results of this embodiment demonstrate that 6 mg / ml collagen bio-ink can be applied directly to the cornea with sufficient adhesion and therefore can be used as a occlusive agent for corneal tissue. For occlusive corneal tissue, a collagen bio-ink with a collagen concentration of 6 mg / ml and 18 mM calcium ions appears to be the optimal composition. The results in Sections 5.3 and 5.4 indicate that 12 mg / ml collagen ink can close larger perforations if there is no leakage. For example, 12 mg / ml collagen ink will be able to close perforations larger than 4 mm in diameter after using other surgical instruments or membranes / films to plug any leakage.

[0346] Example 6: A refined method for generating crosslinkable collagen ink

[0347] Materials and methods

[0348] 6.1 Preparation of Part A and Part B

[0349] Riboflavin powder (Sigma-Aldrich) was added to 5M NaOH at a concentration of 30 mg / ml to form fraction A of the collagen bioink. Ions were added to a 12 mg / ml collagen acetate solution at the concentrations listed in Table 4 to form fraction B of the collagen bioink. After preparation, fractions A and B were mixed in Eppendorf tubes at a ratio of 9:250 to obtain a neutral collagen bioink containing riboflavin, which can be crosslinked.

[0350] 6.2 Storage of Collagen Bio-Ink

[0351] As described in Section 6.1 above, acidic collagen solutions / neutral collagen solutions containing riboflavin (collagen bio-ink) were prepared and stored in a refrigerator (4°C) and a freezer (-20°C). The acidic collagen solutions were stored in 1.5 ml Eppendorf tubes, while the collagen bio-ink was stored in foil-wrapped Eppendorf tubes. Results were made after 1 week, 1 month, 6 months, and 1 year by measuring at 365 nm and 3 mw / cm. 2 To examine the two solutions, neutralize and crosslink them under UV light or crosslink them directly under UV light.

[0352] 6.3 Preparation of Collagen Membranes via Cross-linked Collagen Bio-ink

[0353] A small amount of collagen bio-ink with added riboflavin was transferred onto the surface of a paraffin-coated slide. Two 0-size coverslips, each 0.1 mm thick, were added to either side of the slide, at least 1 cm away from the collagen drop. A 1 mm thick paraffin-coated polypropylene plate was then placed on top of the slide, flattening the collagen drop to 0.1 mm thickness. The solution was then transferred through a 3 mw / cm... 2 UV curing lamp or 10mw / cm 2 The collagen ink was crosslinked using a blue light curing lamp for 3 minutes. After crosslinking, the membrane was removed from the glass slide and washed in PBS. The flexibility and strength of the membrane were observed by rolling and self-expansion tests.

[0354] 6.4 3D Printing Test

[0355] The developed collagen ink was tested using 3D printing. A 3D printer (TRICEP, University of Wollongong) was used. The collagen ink was loaded into a 5mm diameter syringe wrapped in aluminum foil and equipped with a #25 printhead. A printing pressure of 10mw / cm was applied. 2 470nm blue light assists in printing and crosslinking collagen bio-ink. For mesh printing tests, the extrusion rate was set to 0.5mm / min and the printing speed was set to 150mm / min.

[0356] 6.5 Cell Printing Test: Printing corneal-like structures filled with cells.

[0357] The printer used in Section 6.4 above was placed in a sterile bioprinting hood that had undergone 20 minutes of UV sterilization. Collagen ink was prepared using the method described in Section 6.1 above, and Part B was also subjected to 20 minutes of UV sterilization. After mixing Parts A and B, the collagen bioprinting ink was further mixed with 10% FBS DMEM / F12 cell culture medium at a ratio of 9:1, containing 2 million cells / mL, and then loaded into the same syringe used in Section 6.4 above in a sterile hood. The printed structure was cultured in tissue culture medium. Calcein-AM staining was performed 2 weeks post-printing to stain live cells and examine cell viability.

[0358] result

[0359] 6.6 Titration of Parts A and B of Crosslinkable Collagen Bio-ink

[0360] 50, 100, 150, 200, and 250 μL of collagen ink fraction B (acidic collagen ink containing calcium ions) were mixed with specific volumes of collagen ink fraction A to bring the bio-ink to a neutral pH and make it crosslinkable. The amount of fraction A required per volume of fraction B is shown in Table 4. The volume relationship between fraction A and fraction B is linear (…). Figure 28 This indicates that part A and part B have a fixed ratio of 0.036, and can be easily prepared according to this ratio.

[0361] Table 4: Amount of Part A required for each volume of Part B

[0362] Part A Part B 1.8μL 50μL 3.6μL 100μL 5.4μL 150μL 7.2μL 200μL 9.0μL 250μL

[0363] 6.6 Storage of Collagen Bio-Ink

[0364] The results of the storage test are provided in Table 5.

[0365] Table 5: Properties of Collagen Ink Stored Under Different Conditions

[0366]

[0367] 6.8 Preparation of collagen gels of different thicknesses

[0368] A collagen film can be generated by blue light curing. The minimum thickness of the generated collagen gel was measured at 100 μm. The generated collagen gel was visually transparent. The gel could be easily picked up with tweezers without breaking in a folded manner. Figure 29 a). Without additional force, the gel unfolds upon immersion in Milli-Q water. Figure 10 b). The cross-linked collagen structures generated by this method can be up to 4 mm thick. Figure 30 ).

[0369] 6.9 Printing test using a 3D extrusion printer

[0370] Use a printer to print a double-layered 10cm x 10cm mesh as described in section 6.4 above. Figure 31 The lines on the printed mesh are not connected, indicating that the collagen ink is suitable for 3D printing and is compatible with extrusion 3D printers with UV / blue light curing attachments.

[0371] 6.10 Cell Printing Test: Printing corneal-like structures filled with cells.

[0372] The cell-loaded structure was printed using the method described in Section 6.5. After printing, the printed structure was transferred to cell culture medium. Calcein-AM staining showed that the cells remained viable after 2 weeks. Figure 32 ).

Claims

1. A composition comprising: - 3-15 mg / ml of neutral type I collagen; - 0.135-0.3 M NaCl and / or 0.018-0.1 M CaCl2; and - 0.01-0.5% (w / v) riboflavin or 0.01-0.5% (w / v) rose tincture.

2. The composition according to claim 1, wherein the composition comprises fibrinogen and / or thrombin.

3. The composition according to claim 2, wherein the composition comprises 1.6-6 mg / ml of fibrinogen.

4. The composition according to claim 2 or claim 3, wherein the composition comprises 1-5 U / mL of thrombin.

5. The composition according to any one of claims 1 to 4 further comprises one or more of the following: culture medium, hormone, matrix protein, ion source, amino acid, antibiotic, anesthetic, factor XIII, fetal bovine serum (FBS), human serum, platelet lysate.

6. The composition according to any one of claims 1 to 4 further comprises one or more of the following: growth factors, glycoproteins, vitamins, ions other than sodium or calcium ions, fibronectin, and human platelet lysate.

7. The composition according to claim 5 or 6, wherein the composition comprises a culture medium containing the ions and amino acids.

8. The composition according to claim 5, wherein: (i) The matrix protein comprises type IV collagen; and / or (ii) The hormones include insulin.

9. The composition according to claim 6, wherein: (i) The growth factor comprises human epidermal growth factor (hEGF) and / or fibroblast growth factor (FGF); and / or (ii) The vitamins contain ascorbate (vitamin C); and / or (iii) The glycoprotein contains transferrin.

10. The composition according to any one of claims 5 to 7, wherein the ion is a component of an ionic salt included in the composition.

11. The composition according to any one of claims 1 to 10, wherein the composition further comprises mammalian cells.

12. The composition of claim 11, wherein the mammalian cell comprises a human cell.

13. The composition of claim 11, wherein the mammalian cells are composed of human cells.

14. The composition according to any one of claims 1 to 13, wherein the composition comprises: (i) 3-15 mg / ml of neutral type I collagen, 0.135-0.2 M NaCl, and 0.018-0.05 M CaCl2; or (ii) 4-12 mg / ml of neutral type I collagen, 0.135-0.16 M of NaCl, and 0.018-0.03 M of CaCl2; or (iii) 5-11 mg / ml of neutral type I collagen, 0.135-0.14 M of NaCl and 0.018-0.02 M of CaCl2.

15. A method for preparing a composition, the method comprising: (i) Provide a solution comprising: - 3-15 mg / ml of neutral type I collagen; - 0.01-0.5% (w / v) riboflavin or 0.01-0.5% (w / v) rose sulfide; and - 0.135-0.3 M NaCl and / or 0.018-0.1 M CaCl2; (ii) Apply the solution to the surface; and (iii) Apply light to the solution that can activate one or more crosslinking agents.

16. The method of claim 15, wherein the solution is applied to form a surface layer, and wherein steps (ii) and (iii) are repeated multiple times, wherein each layer is applied over the previous layer.

17. The method according to claim 15 or 16, wherein: (a) Combining the riboflavin or rose red with an alkali to form part A; (b) The neutral type I collagen is combined with the NaCl and / or CaCl2 to form part B; as well as (c) Prior to step (iii), mix part A and part B to form a solution.

18. A method for preparing a composition, the method comprising: (i) Provide a solution comprising: - 3-15 mg / ml of neutral type I collagen; - 0.01-0.5% (w / v) riboflavin or 0.01-0.5% (w / v) rose sulfide; and - 0.135-0.3 M NaCl and / or 0.018-0.1 M CaCl2; and (ii) Applying the solution to a surface, wherein the solution is divided into at least two components before being applied to the surface.

19. The method of claim 18, further comprising the step of: (iii) Adding fibrinogen to at least one component to form a formulation (a); (iv) Adding thrombin to at least one component to form a preparation (b); as well as (v) Combine preparations (a) and (b) to form a gel.

20. The method of claim 19, wherein: - In step (iii), 1.6-6 mg / ml of fibrinogen is added to at least one component to form formulation (a); and / or - In step (iv), 1.5 U / mL of thrombin is added to at least one component to form preparation (b).

21. The method according to any one of claims 15 to 20, wherein the solution further comprises one or more of the following: culture medium, hormone, matrix protein, ion source, amino acid, antibiotic, anesthetic, factor XIII, fetal bovine serum (FBS), human serum, platelet lysate.

22. The method according to any one of claims 15 to 20, wherein the solution further comprises one or more of the following: growth factors, glycoproteins, vitamins, ions other than sodium or calcium ions, fibronectin, and human platelet lysate.

23. The method according to claim 21 or 22, wherein the solution comprises a culture medium containing the ions and amino acids.

24. The method of claim 21, wherein: (i) The matrix protein comprises type IV collagen; and / or (ii) The hormones include insulin.

25. The method according to claim 22, wherein: (i) The growth factor comprises human epidermal growth factor (hEGF) and / or fibroblast growth factor (FGF); and / or (ii) The vitamins contain ascorbate (vitamin C); and / or (iii) The glycoprotein contains transferrin.

26. The method according to any one of claims 21 to 23, wherein the ion is a component of an ionic salt included in the mixture.

27. The method according to any one of claims 15 to 26, wherein the solution further comprises mammalian cells.

28. The method of claim 27, wherein the mammalian cell comprises a human cell.

29. The method of claim 27, wherein the mammalian cells are composed of human cells.

30. A composition obtained by or capable of being obtained by the method according to any one of claims 15 to 29.

31. Use of the composition according to any one of claims 1 to 14 or claim 30 in the preparation of a medicament for treating corneal damage.

32. Use of a kit, package, or apparatus comprising neutral type I collagen, NaCl, CaCl2, and riboflavin or rose rosine for preparing a composition comprising: - 3-15 mg / ml of neutral type I collagen; - 0.135-0.3 M NaCl and / or 0.018-0.1 M CaCl2; and - 0.01-0.5% (w / v) riboflavin or 0.01-0.5% (w / v) rose tincture.

33. The use according to claim 32, wherein the composition comprises fibrinogen and thrombin, and wherein... - The fibrinogen is present in the first compartment; - The thrombin is present in the second compartment; and therein - The kit, package, or device is configured to allow separation of the fibrinogen in the first compartment and the thrombin in the second compartment during and after loading the fibrinogen and thrombin into the kit, package, or device, and wherein the kit, package, or device further includes a tool for facilitating mixing of the fibrinogen in the first compartment with the thrombin in the second compartment.

34. The use according to claim 33, wherein the composition comprises: - 1.6-6 mg / ml of fibrinogen; and / or - 1-5 U / mL of thrombin.

35. The use according to any one of claims 32 to 34, wherein the composition further comprises one or more of the following: culture medium, hormone, matrix protein, ion source, amino acid, antibiotic, anesthetic, factor XIII, fetal bovine serum (FBS), human serum, platelet lysate.

36. The use according to any one of claims 32 to 34, wherein the composition further comprises one or more of the following: growth factors, glycoproteins, vitamins, ions other than sodium or calcium ions, fibronectin, and human platelet lysate.

37. The use according to any one of claims 32 to 36, wherein one or more of the neutral type I collagen, NaCl, CaCl2, riboflavin, and rose red are separated from one or more other components in the kit.

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