MODIFIED EXTRACELLULAR MATRIX-BASED HYDROGEL, MANUFACTURING METHOD OF THE SAME AND USE OF THE SAME

Abstract

A modified extracellular matrix-based hydrogel according to an example of the present disclosure includes an extracellular matrix-denatured collagen conjugate formed by a Michael addition reaction between an extracellular matrix having an amine group and a denatured collagen into which an ethylenically unsaturated bond functional group is introduced. The modified extracellular matrix-based hydrogel according to the present disclosure exhibits enhanced mechanical properties (e.g., viscoelasticity) compared to the extracellular matrix hydrogel before modification. In addition, it shows a high cell viability when the bioink is prepared by encapsulating cells in a modified extracellular matrix-based hydrogel according to the present disclosure. In addition, when an artificial living tissue for transplantation (for example, artificial corneal tissue) manufactured by 3-D printing bioink according to the present disclosure is transplanted into a damaged cornea, it can be sutured and has a transparency similar to that of the real cornea, and corneal tissue can be reconstructed without other side effects due to its enhanced mechanical properties. Accordingly, the modified extracellular matrix-based hydrogel according to the present disclosure can be applied in tissue engineering fields and related fields requiring improvement in physical properties and is particularly useful as a material for corneal transplants.

Claims

1. A modified extracellular matrix-based hydrogel comprising an extracellular matrix-denatured collagen conjugate formed by a Michael addition reaction between the extracellular matrix having an amine group and denatured collagen to which an ethylenically unsaturated bond functional group is introduced.

2. The modified extracellular matrix-based hydrogel of claim 1, wherein the extracellular matrix is decellularized extracellular matrix.

3. The modified extracellular matrix-based hydrogel of claim 2, wherein the decellularized extracellular matrix is corneal-derived decellularized extracellular matrix.

4. The modified extracellular matrix-based hydrogel of claim 1, wherein the ethylenically unsaturated bond functional group is selected from the group consisting of a vinyl group, an acryl group and a methacryl group.

5. The modified extracellular matrix-based hydrogel of claim 1, wherein the denatured collagen is selected from methacrylated collagen or acrylated collagen.

6. The modified extracellular matrix-based hydrogel of claim 1, wherein a weight ratio of the extracellular matrix to the denatured collagen contained in the hydrogel is 1:0.05 to 1:0.8.

7. A method for manufacturing the modified extracellular matrix-based hydrogel of claim 1, the method comprising steps of: preparing an extracellular matrix hydrogel having a pH 2 to 5 by dissolving the extracellular matrix having an amine group in an acid solution; forming an extracellular matrix-denatured collagen conjugate by adding a denatured collagen to which an ethylenically unsaturated bond functional group is introduced to the extracellular matrix hydrogel, mixing them uniformly, and inducing a Michael addition reaction; and neutralizing the hydrogel including the extracellular matrix-denatured collagen conjugate to a pH 5.5 to 8.

8. The method for manufacturing modified extracellular matrix-based hydrogel of claim 7, wherein the extracellular matrix is corneal-derived decellularized extracellular matrix.

9. The method for manufacturing modified extracellular matrix-based hydrogel of claim 7, wherein the denatured collagen is selected from methacrylated collagen or acrylated collagen.

10. The method for manufacturing modified extracellular matrix-based hydrogel of claim 7, wherein an addition amount of the denatured collagen is 5 to 80 parts by weight relative to 100 parts by weight of the extracellular matrix contained in the hydrogel.

11. The method for manufacturing modified extracellular matrix-based hydrogel of claim 7, wherein an amount of the extracellular matrix in the extracellular matrix hydrogel is 1 to 4% (w/v).

12. A bioink comprising cells and the modified extracellular matrix-based hydrogel of claim 1, wherein the cells are present in an encapsulated form in the modified extracellular matrix-based hydrogel.

13. The bioink of claim 12, wherein a concentration of cells in the bioink is 1×10.sup.6 cells/ml to 1×10.sup.7 cells/ml.

14. The bioink of claim 12, wherein the cells are corneal-derived cells.

15. An artificial living tissue for transplantation, molded from the bioink of claim 12 by 3-D printing.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1 shows the results of FT-IR analysis of the modified corneal-derived decellularized extracellular matrix hydrogel prepared in an embodiment of the present disclosure.

[0027] FIG. 2 shows the results of analyzing the change in viscoelasticity of the modified corneal-derived decellularized extracellular matrix hydrogel prepared in an embodiment of the present disclosure.

[0028] FIG. 3 shows the results of measuring the cell viability in the bioink prepared in an embodiment of the present disclosure with an optical microscope.

[0029] FIG. 4 shows a slit lamp image and an optical coherence tomography (OCT) image among the in-vivo evaluation results performed in an embodiment of the present disclosure.

[0030] FIG. 5 is a graph showing a behavioral optometry evaluation result among in-vivo evaluation results performed in an embodiment of the present disclosure.

DETAILED DESCRIPTION

[0031] In the following detailed description, reference is made to the accompanying drawing, which forms a part hereof. The illustrative embodiments described in the detailed description, drawing, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented here.

[0032] Hereinafter, the present disclosure is described in detail through embodiments. However, the following embodiments are only for clearly illustrating the technical characteristics of the present disclosure, and do not limit the scope of protection of the present disclosure.

1. Preparation of Corneal-Derived Decellularized Extracellular Matrix (Co-dECM) and Hydrogel Containing the Same

[0033] Corneal-derived decellularized extracellular matrix (Co-dECM) was prepared as follows (See. Kim H, Park M N, Kim J, Jang J, Kim H K and Cho D W 2019 Characterization of cornea-specific bioink: high transparency, improved in vivo safety J. Tissue Eng. 10). First, the entire cornea incised from the calf s eye was washed with a PBS buffer solution containing 100 units/ml of penicillin and 0.1 mg/ml of streptomycin. Thereafter, the epithelium and endothelium were removed from the corneal tissue to obtain a pure corneal stromal layer. Thereafter, the matrix tissue was placed in a 20 mM ammonium hydroxide solution (NH.sub.4OH; 4.98 N aqueous solution) containing 0.5% Triton X-100, and the mixture was stirred for about 4 hours. Thereafter, the matrix tissue was washed with distilled water and treated with a hypotonic Tris hydrochloride (Tris-HCl; pH 7.4) buffer solution for about 24 hours. Thereafter, the matrix tissue was placed in a 10 mM Tris-HCl solution containing 1% (v/v) Triton X-100, and the mixture was stirred at 37° C. for about 24 hours to obtain a corneal-derived decellularized extracellular matrix (Co-dECM) tissue. Thereafter, the corneal decellularized extracellular matrix (Co-dECM) tissue was sterilized by treatment with 1% peracetic acid solution in 50% ethanol for about 10 hours. After completion of the decellularization process, corneal-derived decellularized extracellular matrix (Co-dECM) was freeze-dried overnight and ground into a fine powder using liquid nitrogen and a grinding device. 0.2 g Co-dECM powder was added to 10 ml of acetic acid solution (0.5 M) supplemented with 0.02 g pepsin, and the mixture was uniformly stirred through vortexing, and treated for about 3 days to remove telopeptides in collagen molecules and be completely dissolved to obtain a corneal-derived decellularized extracellular matrix hydrogel having a pH of about 3 to 4 and a corneal-derived decellularized extracellular matrix (Co-dECM) concentration of 2% (w/v). The 2% (w/v) Co-dECM hydrogel was filtered through a 10 μm mesh and stored at 4° C., and it was used for subsequent experiments.

2. Preparation of Modified Corneal-Derived Decellularized Extracellular Matrix (Co-dECM)-Based Hydrogel Using Michael Addition Reaction

Modified Hydrogel Preparation Example 1

[0034] 0.05 g of methacrylated collagen (MAC) was added to 10 ml of corneal-derived decellularized extracellular matrix hydrogel, and the mixture was uniformly stirred through vortexing. While stored at 4° C. for 16 hours, a Michael addition reaction was induced between the methacrylated collagen and the conical-derived decellularized extracellular matrix to modify the corneal-derived decellularized extracellular matrix. Thereafter, a 10 N sodium hydroxide solution was added to the hydrogel to be neutralized to be pH about 7.0 to 7.4 on ice, thereby obtaining a modified corneal-derived decellularized extracellular matrix-based hydrogel.

Modified Hydrogel Preparation Example 2

[0035] Except that 0.01 g of methacrylated collagen (MAC) was added to 10 ml of corneal-derived decellularized extracellular matrix hydrogel, the modified corneal-derived extracellular matrix-based hydrogel was obtained under the same conditions and in the same manner as in Preparation Example 1.

Modified Hydrogel Preparation Example 3

[0036] Except that 0.1 g of methacrylated collagen (MAC) was added to 10 ml of corneal-derived decellularized extracellular matrix hydrogel, the modified corneal-derived extracellular matrix-based hydrogel was obtained under the same conditions and in the same manner as in Preparation Example 1.

3. Characterization of Modified Hydrogels

(1) Analysis of Chemical Changes in Modified Hydrogels

[0037] Chemical changes of the modified corneal-derived decellularized extracellular matrix-based hydrogel prepared in Modified Hydrogel Preparation Examples 1 to 3 were measured through FT-IR analysis.

[0038] FIG. 1 shows the results of FT-IR analysis of the modified corneal-derived decellularized extracellular matrix hydrogel prepared in an embodiment of the present disclosure. In FIG. 1, the term “Co-dECM” refers to corneal-derived decellularized extracellular matrix hydrogel before modification, and “MAC” refers to methacrylated collagen. In addition, in FIG. 1, the term “0.5MACCO” refers to the modified corneal-derived decellularized extracellular matrix-based hydrogel prepared in Modified Hydrogel Preparation Example 1, and the term “0.1MACCO” refers to the modified corneal-derived decellularized extracellular matrix-based hydrogel prepared in Modified Hydrogel Preparation Example 2, and the term “1.0MACCO” refers to the modified corneal-derived decellularized extracellular matrix-based hydrogel prepared in Modified Hydrogel Preparation Example 3.

[0039] In addition, the values of peaks corresponding to C—C single bonds and C═C double bonds among the FT-IR analysis results of the modified corneal-derived decellularized extracellular matrix hydrogel prepared in embodiments of the present disclosure are summarized in Table 1 below.

TABLE-US-00001 TABLE 1 Classi- fication MAC 1.0MACCO 0.5MACCO 0.1MACCO Co-dECM C═C −17.47 −2.92 −2.40 −1.87 −1.46 peak value C—C −2.44 −5.81 −5.83 −5.42 −5.06 peak value

[0040] In the modified hydrogel Preparation Examples 1 to 3, the Michael addition reaction between the methacrylated collagen and the corneal-derived decellularized extracellular matrix proceeds by the following mechanism.

Mechanism of Michael Addition Reaction Between Methacrylated Collagen and Corneal-Derived Decellularized Extracellular Matrix

[0041] ##STR00001##

[0042] In the corneal-derived decellularized extracellular matrix hydrogel having a pH of about 3 to 4, the amine group present in the corneal-derived decellularized extracellular matrix has high reactivity to lose protons easily, and ethylenically unsaturated bonds present in methacrylated collagen obtains a proton source. Thereafter, it forms a single bond with an amine group present in the corneal-derived decellularized extracellular matrix.

[0043] 0.1% (w/v), 0.5% (w/v) and 1.0% (w/v) methacrylated collagens (MAC) were added to corneal-derived decellularized extracellular matrix (Co-dECM) hydrogel, respectively, and Michael addition reaction was induced to change the amount of C—C single bond and C═C double bond. As shown in FIG. 1 and Table 1, methacrylated collagen (MAC) contains methacrylic groups and thus has the most C═C double bonds, whereas corneal-derived decellularized extracellular matrix (Co-dECM) had the fewest C═C double bonds. When methacrylated collagen (MAC) reacts with corneal-derived decellularized extracellular matrix (Co-dECM), the C═C double bond is reduced, and C—C single bond is increased compared to methacrylated collagen (MAC), indicating Michael addition reaction proceeded.

[0044] In the case of 0.1MACCO hydrogel, C—C single bonds and C═C double bonds were increased compared to Co-dECM hydrogel. In the case of 0.5MACCO hydrogel, C—C single bonds and C═C double bonds were increased compared to Co-dECM hydrogel, and C—C single bonds and C═C double bonds were also increased compared to 0.1MACCO hydrogel. These results indicate that the reaction between Co-dECM and MAC proceeded more in the case of the 0.5MACCO hydrogel compared to the 0.1MACCO hydrogel. Meanwhile, in the case of 1.0MACCO hydrogel, C—C single bonds and C═C double bonds were increased compared to Co-dECM hydrogel, but C—C single bonds were almost the same compared to 0.5MACCO hydrogel. These results indicate that the reaction is closest to the maximum in the 0.5 MACCO hydrogel.

[0045] From the results of FIG. 1 and Table 1, the present inventors found that the concentration of methacrylated collagen (MAC) for the modification of corneal-derived decellularized extracellular matrix (Co-dECM) hydrogel was 0.5% (w/v), and the modified corneal-derived decellularized extracellular matrix-based hydrogel prepared in Modified Hydrogel Preparation Example 1 was used in the subsequent preparation experiments of bioink and artificial corneal tissue for transplantation.

(2) Analysis of Changes in Viscoelasticity of Modified Hydrogels

[0046] In order to analyze the change in viscoelasticity of the modified hydrogel, the rheological properties were measured using an advanced hybrid rheometer equipped with a 25 mm diameter plate. First, the normal shear sweep analysis of the hydrogel was performed at 4° C., a temperature condition generally used in the biofabrication process, and specifically, the viscosity according to the shear rate was measured. Further, the time sweep analysis of the hydrogel was performed at 37° C., a temperature condition used after the biofabrication process, and specifically, the complex modulus (G*) at 2% strain over time was measured. The time sweep analysis is used to study the gelation kinetics of hydrogels. FIG. 2 shows the results of analyzing the change in viscoelasticity of the modified corneal-derived decellularized extracellular matrix hydrogel prepared in an embodiment of the present disclosure. The graph on the left of FIG. 2 shows a normal shear sweep analysis result, and the graph on the right shows a time sweep analysis. In addition, in FIG. 2, the term “Co-dECM” refers to the corneal-derived decellularized extracellular matrix hydrogel before modification, and “0.5MACCO” refers to the modified corneal-derived extracellular matrix-based hydrogel prepared in Modified Hydrogel Preparation Example 1. As shown in the normal shear sweep analysis result of FIG. 2, it was confirmed that both the corneal-derived decellularized extracellular matrix hydrogel before modification and the modified corneal-derived extracellular matrix-based hydrogel prepared in Preparation Example 1 have shear thinning properties, and there was no significant difference between the two materials. However, as shown in the time sweep analysis result of FIG. 2, the complex modulus (G*) value of the modified corneal-derived extracellular matrix-based hydrogel prepared in Preparation Example 1 was approximately 78 times greater than the corneal-derived decellularized extracellular matrix hydrogel before modification after about 30 minutes. This result means that the effect of the Michael addition reaction appears in the gelation process of the hydrogel.

4. Preparation of Bioink and Artificial Corneal Tissue for Transplantation

Bioink Preparation Example 1

[0047] A bioink was prepared by encapsulating the differentiated keratocytes in the modified corneal-derived decellularized extracellular matrix-based hydrogel prepared in Preparation Example 1.

[0048] Differentiated keratocytes were prepared as follows (See. [Park M N, Kim B, Kim H, Park S H, Lim M H, Choi Y J, Yi H G, Jang J, Kim S W and Cho D W 2017 Human turbinate-derived mesenchymal stem cells differentiated into keratocyte progenitor cells J. Clin. Exp. Ophthalmol. 8 627]. Human turbinate derived mesenchymal stem cells (hTMSCs; obtained from Catholic University of Korea, St. Mary's Hospital) were placed in normal DMEM (Dulbecco's Modified Eagle's Medium) containing 10% (v/v) fetal bovine serum and 1% (v/v) penicillin and cultured in a humidified 5% carbon dioxide atmosphere and a temperature of 37° C. Then, in the second passage, the normal medium was replaced with a differentiation medium containing 10 ng/ml KGF/EGF and cultured for one day to obtain differentiated keratocytes. Thereafter, the keratocytes obtained from the second or third passage were encapsulated at a concentration of 5×10.sup.6 cells/ml in the modified corneal-derived extracellular matrix-based hydrogel prepared in Preparation Example 1 to prepare the bioink in the form of the corneal decellularized extracellular matrix (Co-dECM) hydrogel in which the cells were encapsulated. The cell encapsulation process was performed on ice. In addition, an artificial corneal tissue having a diameter of 4 mm and a height of 200 μm was produced from the bioink using a 3-dimensional cell printing system.

Bioink Preparation Example 2

[0049] Except that differentiated keratocytes were encapsulated in a corneal-derived decellularized extracellular matrix hydrogel before modification, a bioink was prepared, and an artificial corneal tissue was produced under the same conditions and in the same manner as in Bioink Preparation Example 1.

5. Cell Viability Analysis

[0050] FIG. 3 shows the results of measuring the cell viability in the bioink prepared in an embodiment of the present disclosure with an optical microscope. In FIG. 3, the term “0.5MACCO” refers to the bioink prepared in Bioink Preparation Example 1, and the term “Co-dECM” refers to the bioink prepared in Bioink Preparation Example 2. As shown in FIG. 3, both the case of using the modified corneal-derived extracellular matrix-based hydrogel prepared in Preparation Example 1 as a hydrogel for encapsulating cells and the case of using the corneal-derived decellularized extracellular matrix hydrogel before modification showed 95% or more cell viability.

6. In-vivo Evaluation

[0051] In order to observe the in-vivo compatibility and the level of vision recovery after transplantation of the artificial corneal tissue prepared in Bioink Preparation Example 1, animal experiments were performed in Daegu-Gyeongbuk Advanced Medical Industry Promotion Foundation (DGMIF, the approval number of protocol: DGMIF-17080801-00) according to the ARVO statement on the use of animals by Ophthalmic and Vision Research. Eight healthy beagle dogs (8 weeks old male having an average weight of about 4 kg) were anesthetized with 30 mg/ml of ketamine and 10 mg/ml of rompun. A 3-quarter annular incision with a diameter of 5 mm was made using a crescent knife. Thereafter, the beagles with corneal scratches were divided into two groups, such as a negative control group and an experimental group. No separate treatments were taken for the beagle corresponding to the negative control group. The artificial corneal tissue prepared in Bioink Preparation Example 1 was transplanted on the corneal matrix of the beagles corresponding to the experimental group. Meanwhile, in the case of transplanting the artificial corneal tissue prepared in Bioink Preparation Example 2, the incision site was not smoothly sutured as a result of the pre-test, so it was not included in the experimental group. Then, the incision site was sutured with 10-0 ethilon nylond, and the operated eye was treated with eye drops containing olymyxin B, neomycin and dexamethasone (Forus, Samil Pharm. Co., Ltd, Korea) once a day for 2 weeks.

[0052] For the experimental animals, slit lamp examination and OCT examination were performed at predetermined time intervals for 24 weeks after transplantation. Further, behavioral optometry was evaluated in a state in which only the incised eye was exposed for visual acuity test 5 weeks after transplantation of the experimental animals. The behavioral optometry evaluation was performed by the veterinarian, which is one of the animal visual acuity evaluations that indirectly evaluate visual acuity by evaluating whether it avoids obstacles and whether it can follow moving objects with its eyes, with a full score of 5.

[0053] FIG. 4 shows a slit lamp image and an optical coherence tomography (OCT) image among the in-vivo evaluation results performed in an embodiment of the present disclosure. As shown in FIG. 4, in the case of the negative control group, as time passed, corneal edema occurred entirely in addition to the scratched area in the cornea, and the cornea became cloudy. On the other hand, in the case of the experimental group, not only could the cornea be sutured, but the transparency gradually recovered as time passed. FIG. 5 is a graph showing a behavioral optometry evaluation result among in-vivo evaluation results performed in an embodiment of the present disclosure. As shown in FIG. 5, the negative control group showed a significant decrease in visual acuity, while the experimental group recorded a value close to 5, the perfect score for behavioral optometry, indicating that visual acuity was greatly recovered. These results indicate that in the case of the artificial corneal tissue prepared in Bioink Preparation Example 1, it is possible to suture the incised cornea and greatly help in corneal reconstruction, such as tolerating internal intraocular pressure well and recovering visual acuity even after transplantation.

[0054] As described above, the present disclosure has been described through the above embodiments, but the protection scope of the present disclosure is not necessarily limited thereto, and various modifications can be made within the scope and spirit of the present disclosure. Accordingly, the protection scope of the present disclosure should not be limited to the specific embodiments disclosed as the best mode but should be construed to include all embodiments falling within the scope of the claims appended to the present disclosure.

[0055] From the foregoing, it will be appreciated that various embodiments of the present disclosure have been described herein for purposes of illustration, and that various modifications may be made without departing from the scope and spirit of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting, with the true scope and spirit being indicated by the following claims.