EXTRACELLULAR MATRIX-BASED BIOADHESIVE

Abstract

An embodiment of the present disclosure provides an extracellular matrix-based bioadhesive as an adhesive in the form of a composition including an extracellular matrix-containing hydrogel and a gelatin curing agent, wherein the extracellular matrix-containing hydrogel is gelatinized. Since the extracellular matrix-based bioadhesive according to an embodiment of the present disclosure has the same or similar rheological properties as gelatin, the bioadhesive has flowability at a temperature of 30° C. or higher and may be evenly and easily applied to a lesion site in the body. In addition, the extracellular matrix-based bioadhesive according to an embodiment of the present disclosure may adhere well to the lesion site because of a level of adhesive strength that is about 2 to 6 times higher than that of fibrin glue used as a commercial tissue adhesive. In addition, the extracellular matrix-based bioadhesive according to an embodiment of the present disclosure is based on a tissue-derived extracellular matrix, and thus includes a tissue-derived wound healing component or a tissue regeneration component, and may be used for wound healing or tissue regeneration in addition to bioadhesive applications.

Claims

1. An extracellular matrix-based bioadhesive as an adhesive in the form of a composition including an extracellular matrix-containing hydrogel and a gelatin curing agent, wherein the extracellular matrix-containing hydrogel is gelatinized.

2. The extracellular matrix-based bioadhesive of claim 1, wherein the extracellular matrix is a decellularized extracellular matrix.

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

4. The extracellular matrix-based bioadhesive of claim 1, wherein the extracellular matrix-containing hydrogel is gelatinized by thermal denaturation of collagen, which is a component of an extracellular matrix.

5. The extracellular matrix-based bioadhesive of claim 1, wherein the gelatin curing agent is selected from a combination of ruthenium and sodium persulfate or riboflavin.

6. The extracellular matrix-based bioadhesive of claim 5, wherein: the gelatin curing agent is a combination of ruthenium and sodium persulfate; a concentration of ruthenium in the bioadhesive is 0.1 to 2 mM; and a concentration of sodium persulfate is 1 to 20 mM.

7. A method for manufacturing an extracellular matrix-based bioadhesive, the method including: preparing an extracellular matrix-containing hydrogel neutralized by adjusting a pH of the extracellular matrix-containing hydrogel to 6 to 8.5; heat-treating the neutralized extracellular matrix-containing hydrogel at a temperature of 50 to 60° C. to denature collagen, which is a component of an extracellular matrix, to obtain a gelatinized extracellular matrix-containing hydrogel; and adding and mixing a gelatin curing agent to the gelatinized extracellular matrix-containing hydrogel to obtain a bioadhesive.

8. The method for manufacturing an extracellular matrix-based bioadhesive of claim 7, wherein the extracellular matrix is a decellularized extracellular matrix.

9. The method for manufacturing an extracellular matrix-based bioadhesive of claim 8, wherein the decellularized extracellular matrix is a corneal-derived decellularized extracellular matrix.

10. The method for manufacturing an extracellular matrix-based bioadhesive of claim 7, wherein a content of the extracellular matrix in the extracellular matrix-containing hydrogel is 1 to 4% (w/v).

11. The method for manufacturing an extracellular matrix-based bioadhesive of claim 7, wherein a heat treatment time of the neutralized extracellular matrix-containing hydrogel is 10 to 40 minutes.

12. The method for manufacturing an extracellular matrix-based bioadhesive of claim 7, wherein the gelatin curing agent is added when a temperature of the gelatinized extracellular matrix-containing hydrogel is 30 to 45° C.

13. The method for manufacturing an extracellular matrix-based bioadhesive of claim 7, wherein the gelatin curing agent is selected from a combination of ruthenium and sodium persulfate or riboflavin.

14. The method for manufacturing an extracellular matrix-based bioadhesive of claim 7, wherein: the gelatin curing agent is a combination of ruthenium and sodium persulfate; a concentration of ruthenium in the bioadhesive is 0.1 to 2 mM; and a concentration of sodium persulfate is 1 to 20 mM.

15. A composition for wound healing or tissue regeneration including the extracellular matrix-based bioadhesive of claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 is a graph showing the results of time sweep analysis of the neutralized corneal-derived decellularized extracellular matrix (Co-dECM) hydrogels prepared in an example embodiment of the present disclosure (black line: temperature profile; gray line: loss tangent [Tan(delta)]).

[0024] FIG. 2 is a graph showing the results of analyzing the adhesive ability of the extracellular matrix-based bioadhesive prepared in an example embodiment of the present disclosure. In FIG. 2, the term “dECM” refers to a neutralized Co-dECM hydrogel, the term “0.5/5 mM” refers to the extracellular matrix-based bioadhesive prepared in Preparation Example 1, and the term “1/10 mM” refers to the extracellular matrix-based bioadhesive prepared in Preparation Example 2.

[0025] FIG. 3 is an image of measuring the cytotoxicity of the extracellular matrix-based bioadhesive prepared in an example embodiment of the present disclosure. In FIG. 3, the term “Control” on the left refers to a neutralized Co-dECM hydrogel, and the term “Vis+Ru/SPS” on the right refers to the extracellular matrix-based bioadhesive prepared in Preparation Example 2.

[0026] FIG. 4 is an image showing the process and results of applying the extracellular matrix-based bioadhesive prepared in Preparation Example 1 in vitro of example embodiments of the present disclosure.

DETAILED DESCRIPTION

[0027] 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.

[0028] Hereinafter, the present disclosure will be described in more detail through example embodiments. However, the following example embodiments are only for clearly illustrating the technical features of the present disclosure, and do not limit the scope of right of the present disclosure.

[0029] 1. Preparation of Corneal-Derived Decellularized Extracellular Matrix (Co-dECM) and Hydrogel Containing the Same

[0030] 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 pig'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 used for subsequent experiments.

[0031] 2. Preparation of Extracellular Matrix-Based Bioadhesive

[0032] Preparation Example 1.

[0033] A 10 N concentration of sodium hydroxide solution was added to the 2% (w/v) Co-dECM hydrogel and stirred to be neutralized to a pH of 7.0 to 7.4. Thereafter, the neutralized Co-dECM hydrogel was heated to 55° C., maintained for 20 minutes, and thermal denaturalized, thereby obtaining the gelatinized Co-dECM hydrogel. Then, when the gelatinized Co-dECM hydrogel was cooled slowly at room temperature to about 37° C., photocuring agents ruthenium and sodium persulfate were added so that the final concentrations became 0.5 mM and 5 mM, respectively, and stirred to prepare a bioadhesive. All solvents of the ruthenium solution and the sodium persulfate solution were Dulbecco's phosphate-buffered saline (DPBS). When the bioadhesive is irradiated with a visible light having a wavelength of about 400 to 450 nm, the tyrosine residues present in the gelatinized Co-dECM hydrogel are oxidized and converted into tyrosine free radicals, and curing is promoted by forming a di-tyrosine covalent bond with a nearby tyrosine residue.

[0034] Preparation Example 2.

[0035] A 10 N concentration of sodium hydroxide solution was added to the 2% (w/v) Co-dECM hydrogel and stirred to be neutralized to a pH of 7.0 to 7.4. Thereafter, the neutralized Co-dECM hydrogel was heated to 55° C., maintained for 20 minutes, and thermal denaturalized, thereby obtaining the gelatinized Co-dECM hydrogel. Then, when the gelatinized Co-dECM hydrogel was cooled slowly at room temperature to about 37° C., photocuring agents ruthenium and sodium persulfate were added so that the final concentrations became 1 mM and 10 mM, respectively, and stirred to prepare a bioadhesive. All solvents of the ruthenium solution and the sodium persulfate solution were Dulbecco's phosphate-buffered saline (DPBS).

[0036] 3. Observation of Phase Change of Co-dECM Hydrogel during Thermal Denaturation Process

[0037] A 10 N concentration of sodium hydroxide solution was added to the 2% (w/v) Co-dECM hydrogel and stirred to be neutralized to a pH of 7.0 to 7.4. Thereafter, the phase change according to the heat treatment of the neutralized Co-dECM hydrogel was measured using an advanced hybrid rheometer equipped with a 25 mm diameter plate. Specifically, the time sweep analysis was performed while applying a predetermined temperature profile over time to the neutralized Co-dECM hydrogel, and Tan (delta) at 10% strain was measured. FIG. 1 is a graph showing the results of time sweep analysis of the neutralized corneal-derived decellularized extracellular matrix (Co-dECM) hydrogels prepared in an example embodiment of the present disclosure (black line: temperature profile; gray line: loss tangent [Tan(delta)]). As shown in FIG. 1, when the neutralized Co-dECM hydrogel was heated to a preset temperature profile, a total of three phase changes occurred. Specifically, when the neutralized Co-dECM hydrogel was heated from 4° C. to 37° C., a primary phase change occurred at 37° C. The primary phase change means that the Co-dECM hydrogel, of which major component is collagen, is cured. Thereafter, the temperature condition was maintained at 37° C. for about 3,000 seconds to stabilize the gelation deformation, and then, the Co-dECM hydrogel was heated to 55° C., at which time a secondary phase change occurred. The secondary phase change means that collagen, which is a major component of the Co-dECM hydrogel, is gelatinized through thermal denaturation after curing. Thereafter, the temperature condition was maintained at 55° C. for about 1,200 seconds to stabilize the thermal denaturation process, and then the Co-dECM hydrogel was cooled to 4° C., at which time a tertiary phase change occurred. The tertiary phase change means that the gelatinized Co-dECM hydrogel is solidified according to temperature in accordance with the properties of gelatin.

[0038] 4. Component Analysis of Gelatinized Corneal-Derived Decellularized Extracellular Matrix (Co-dECM) Hydrogel

[0039] A 10 N concentration of sodium hydroxide solution was added to the 2% (w/v) Co-dECM hydrogel and stirred to be neutralized to a pH of 7.0 to 7.4. Thereafter, the neutralized Co-dECM hydrogel was heated to 55° C., maintained for 20 minutes, and thermal denaturalized, thereby obtaining the gelatinized Co-dECM hydrogel. Thereafter, the gelatinized Co-dECM hydrogel was freeze-dried and then pulverized to make powder, and the components were analyzed using a proteomics analysis method. In Table 1 below, 15 major components, which are the results of proteomics analysis of the gelatinized Co-dECM hydrogel, are summarized.

TABLE-US-00001 TABLE 1 Component content Matrisome Description of Components (weight %) Collagens collagen alpha-1 (I) chain isoform 44.34 X1 [Sus scrofa] Collagens collagen alpha-2 (I) chain precursor 26.71 [Sus scrofa] Collagens collagen alpha-1 (II) chain [Sus scrofa] 8.94 Collagens collagen alpha-3 (VI) chain [Sus scrofa] 6.70 Collagens collagen alpha-1 (XII) chain isoform 0.66 X1 [Sus scrofa] Collagens collagen alpha-2 (VI) chain [Sus scrofa] 0.61 Collagens collagen alpha-2 (V) chain precursor 0.56 [Sus scrofa] ECM thrombospondin type-1 domain-containing 0.22 Glycoproteins protein 4 isoform X1 [Sus scrofa] ECM pigment epithelium-derived factor isoform 0.20 Regulators X1 [Sus scrofa] Proteoglycans decorin precursor [Sus scrofa] 0.20 Proteoglycans keratocan [Sus scrofa] 0.18 Proteoglycans aldehyde dehydrogenase, dimeric NADP- 0.17 preferring [Sus scrofa] Collagens collagen alpha-1 (V) chain precursor 0.16 [Sus scrofa] Collagens collagen alpha-1 (XI) chain [Sus scrofa] 0.07 ECM transforming growth factor-beta-induced 0.06 Glycoproteins protein ig-h3 [Sus scrofa]

[0040] As shown in Table 1 above, the gelatinized Co-dECM hydrogel was identified to contain the most collagen component, and in addition, transforming growth factor-beta-induced protein, which is a major component of corneal wound healing; decorin, a major component of transparent corneal regeneration; and keratocan, aldehyde dehydrogenase, etc., which actually control corneal function, were identified to be contained.

[0041] 5. Analysis of Adhesive Ability of Extracellular Matrix-Based Bioadhesive

[0042] After heating the extracellular matrix-based bioadhesives prepared in Preparation Examples 1 and 2 to about 30° C. to have flowability, 10 ml thereof per test was applied to a measurement reservoir provided in a texture analyzer and irradiated with blue light at an intensity of 10 mW/cm.sup.2 for about 10 minutes to cure the bioadhesives. Then, the adhesive ability of the cured bioadhesives was measured. In addition, as a control group, the adhesive ability was measured after applying and curing the Co-dECM hydrogel neutralized to a pH of 7.0 to 7.4 in the same manner without the addition of a curing agent and without undergoing a thermal denaturation process. FIG. 2 is a graph showing the results of analyzing the adhesive ability of the extracellular matrix-based bioadhesive prepared in an example embodiment of the present disclosure. In FIG. 2, the term “dECM” refers to a neutralized Co-dECM hydrogel, the term “0.5/5 mM” refers to the extracellular matrix-based bioadhesive prepared in Preparation Example 1, and the term “1/10 mM” refers to the extracellular matrix-based bioadhesive prepared in Preparation Example 2. As shown in FIG. 2, as the content of the photocuring agent in the bioadhesive increased, the adhesive ability of the bioadhesive was improved. The adhesive ability of the extracellular matrix-based bioadhesives prepared in Preparation Examples 1 and 2 is 2 to 6 times higher than the adhesive strength of about 1.2 N.Math.mm of fibrin glue used as a commercial tissue adhesive.

[0043] 6. Cytotoxicity Evaluation of Extracellular Matrix-Based Bioadhesives

[0044] 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 Dulbecco's Modified Eagle's Medium (DMEM) containing 10% (v/v) fetal bovine serum and 1% (v/v) penicillin/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, after applying the differentiated keratocytes obtained on the extracellular matrix-based bioadhesive prepared in Preparation Example 2, the cells were cultured for 4 days and the cell viability was observed to evaluate the cytotoxicity of the extracellular matrix-based bioadhesive. In addition, as a control group, the cell viability was observed after merely applying and culturing the cells in the same manner on the Co-dECM hydrogel neutralized to a pH of 7.0 to 7.4 without the addition of a curing agent and without undergoing a thermal denaturation process. FIG. 3 is an image of measuring the cytotoxicity of the extracellular matrix-based bioadhesive prepared in an example embodiment of the present disclosure. In FIG. 3, the term “Control” on the left refers to a neutralized Co-dECM hydrogel, and the term “Vis+Ru/SPS” on the right refers to the extracellular matrix-based bioadhesive prepared in Preparation Example 2. As shown in FIG. 3, both the neutralized Co-dECM hydrogel and the extracellular matrix-based bioadhesive prepared in Preparation Example 2 showed a cell viability of 95% or more, which was verified to have little cytotoxicity.

[0045] 7. In Vitro Application of Extracellular Matrix-Based Bioadhesive

[0046] A 350 μm deep defect was made in the pig's eyes, and the extracellular matrix-based bioadhesive prepared in Preparation Example 1 was applied to the defected area. Thereafter, the bioadhesive was cured by irradiating blue light with a wavelength of 400 to 450 nm to the applied bioadhesive for about 3 minutes using a diagnostic device actually used for the evaluation of macular degeneration in ophthalmology. FIG. 4 is an image showing the process and results of applying the extracellular matrix-based bioadhesive prepared in Preparation Example 1 in vitro of example embodiments of the present disclosure. After curing the bioadhesive, it was identified that the bioadhesive did not fall off and adhered well to the corneal tissue of the pig even when pressure was applied to the pig's eyes by hand or a force was applied to a specific area with tweezers.

[0047] As described above, the present disclosure has been described through the above example embodiments, but the present disclosure is not necessarily limited thereto, and various modifications may be made without departing from the scope and spirit of the present disclosure. Accordingly the scope of protection of the present disclosure should be construed to include all embodiments falling within the scope of the claims appended to the present disclosure.

[0048] 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.