HYDROGEL PATCH

Abstract

Provided are a hydrogel patch, a method of preparing the same, and a composition for the treatment of spinal cord injury including the hydrogel patch. The hydrogel patch enables a spinal cord injury patient to be treated in a manner which is non-invasive to the spinal cord.

Claims

1. A hydrogel patch comprising: at least one selected from fibrin and fibrinogen; laminin; and hyaluronic acid or a salt thereof.

2. The hydrogel patch of claim 1, further comprising a cell growth factor.

3. The hydrogel patch of claim 2, wherein the cell growth factor comprises a neuronal cell growth factor, a vascular endothelial cell growth factor, a fibroblast growth factor, a bone morphogenetic protein, an epidermal growth factor, a hepatocyte growth factor, a transforming growth factor, or a combination thereof.

4. The hydrogel patch of claim 3, wherein the neuronal growth factor comprises at least one selected from brain-derived neurotrophic factor (BDNF), a glial cell-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), basic fibroblast growth factor (bFGF), cyclic adenosine monophosphate (cAMP), a neurotrophin (NT), neurotropin-3 (NT3), neurotropin-4 (NT4), triiodo-L-thyronine (T3), sonic hedgehog (SHH), and platelet-derived growth factor (PDGF).

5. The hydrogel patch of claim 3, wherein the vascular endothelial cell growth factor is vascular endothelial growth factor (VEGF).

6. The hydrogel patch of claim 1, wherein the hydrogel patch does not include cells.

7. The hydrogel patch of claim 1, wherein the hydrogel patch has a porous surface.

8. The hydrogel patch of claim 1, wherein the hydrogel patch undergoes a reversible phase transition into a solid state, a semi-solid state, or a liquid state, in accordance with temperature.

9. The hydrogel patch of claim 1, wherein a shape of the hydrogel patch conforms to a shape of an injured tissue site when the hydrogel patch is applied onto the injured tissue site.

10. The hydrogel patch of claim 1, wherein the hydrogel patch is used for regenerating or covering an injured tissue.

11. (canceled)

12. (canceled)

13. The hydrogel patch of claim 1, wherein, in the hydrogel patch, a concentration of fibrin or fibrinogen is in a range of 0.5 mg/ml to 20 mg/ml, a concentration of laminin is in a range of 1 g/ml to 100 g/ml, or a concentration of hyaluronic acid or a salt thereof is in a range of 10 g/ml to 5 mg/ml.

14. (canceled)

15. The hydrogel patch of claim 1, further comprising thrombin.

16. A method of treating a spinal cord injury (SCI), the method including administering a hydrogel patch comprising at least one selected from fibrin and fibrinogen; laminin; and hyaluronic acid or a salt thereof to a subject in need thereof.

17. The method of claim 16, further comprising a cell growth factor.

18. (canceled)

19. (canceled)

20. The method of claim 16, wherein when the pharmaceutical composition is solid or semi-solid, the pharmaceutical composition has a porous surface.

21. The method of claim 16, wherein the pharmaceutical composition has the form of a powder.

22. (canceled)

23. The method of claim 16, wherein, in the hydrogel patch, a concentration of fibrin or fibrinogen is in a range of 0.5 mg/ml to 20 mg/ml, a concentration of laminin is in a range of 1 g/ml to 100 g/ml, or a concentration of hyaluronic acid is in a range of 10 g/ml to 5 mg/ml.

24. The method of claim 16, further comprising thrombin.

25. The method of claim 16, wherein the spinal cord injury is a chronic spinal cord injury.

26. A method of preparing a hydrogel patch, the method comprising adding thrombin to a sol-phase composition comprising fibrinogen, laminin, and hyaluronic acid or a pharmaceutically acceptable salt thereof, wherein the hydrogel patch is low-temperature preserved or cryopreserved in a solution at a temperature of 4 C. to 210 C.

27. (canceled)

28. (canceled)

Description

BRIEF DESCRIPTION OF DRAWINGS

[0061] FIG. 1 is a view illustrating a hydrogel patch preparation method according to an embodiment and a hydrogel patch having a size of 0.3 mm to 0.8 mm.

[0062] FIG. 2 shows photographs illustrating a hydrogel patch formulation (liquid, gel, powder) (top panels, left to right) and gelation (bottom panels) of a powder formulation according to an embodiment.

[0063] FIG. 3 shows scanning electron microscope (SEM) images of a cross-section and a surface of a hydrogel patch according to an embodiment before and after cryopreservation thereof.

[0064] FIG. 4 shows effects of the composition (fibrin, hyaluronic acid, and laminin) of a hydrogel patch according to an embodiment on mouse neural stem cell differentiation in vitro, as confirmed by optical microscopy and immunofluorescence staining, compared with when the composition is not used (bare); the scale bars represent 100 m and 50 m, respectively.

[0065] FIG. 5 shows immunofluorescence staining images of distribution of neuronal (Tuj1) and astrocytic (GFAP) differentiation, showing effects of a hydrogel patch according to an embodiment on mouse neural stem cell differentiation in vitro, when the hydrogel patch includes each component alone and when the hydrogel patch includes a combination of the two components; the scale bar represents 50 m.

[0066] FIG. 6 shows a neuronal (Tuj1) differentiation distribution graph which is used to quantify effects of a hydrogel patch according to an embodiment on mouse neural stem cell differentiation in vitro, when the hydrogel patch includes each component alone and when the hydrogel patch includes a combination of the two components; *p<0.05, **p<0.005, and ***p<0.0005.

[0067] FIG. 7 shows a neuronal (Tuj1) differentiation distribution graph which is used to quantify effects of a hydrogel patch according to an embodiment on mouse neural stem cell differentiation in vitro, when a growth factor is added to the hydrogel patch including each component alone and the hydrogel patch including a combination of the two components; *p<0.05, **p<0.005, and ***p<0.0005.

[0068] FIG. 8 shows a neuronal (Tuj1) differentiation distribution graph which is used to quantify effects of a hydrogel patch according to an embodiment on mouse neural stem cell differentiation in vitro, when a growth factor (FGF, BDNF, GDNF, cAMP, NT3, PDGF-AA, SHH, T3, or VEGF) is added to the hydrogel patch; *p<0.05, **p<0.005, and ***p<0.0005.

[0069] FIG. 9 confirms biodegradation of a hydrogel patch according to an embodiment when the hydrogel patch is transplanted in vivo.

[0070] FIG. 10 shows images illustrating a process of transplanting a hydrogel patch according to an embodiment into an injured spinal cord.

[0071] FIG. 11 shows images of a spinal cord injury (SCI) animal model for observation of the movement of hind legs thereof, one week after SCI.

[0072] FIG. 12 shows an image of an SCI animal model for observation of the movement of hind legs thereof, eight weeks after SCI, when PBS alone, without the hydrogel patch, is applied to the transplantation site.

[0073] FIG. 13 shows an image of an SCI animal model for observation of the movement of hind legs thereof, eight weeks after SCI, when hydrogel patch 2 (hydrogel patch 2 without GF) is transplanted.

[0074] FIG. 14 shows an image of an SCI animal model for observation of the movement of hind legs thereof, eight weeks after SCI, when hydrogel patch 5 (hydrogel patch 2 with GF) is transplanted.

[0075] FIG. 15 shows BBB scores obtained according to transplantation of a hydrogel patch according to an embodiment (hydrogel patch 3 with PBS, hydrogel patch 3, and hydrogel patch 6).

[0076] FIG. 16 shows BBB scores obtained according to transplantation of a hydrogel patch according to an embodiment (hydrogel patch 1, hydrogel patch 2, and hydrogel patch 3).

[0077] FIG. 17 shows BBB scores obtained according to transplantation of a hydrogel patch according to an embodiment (hydrogel patch 4, hydrogel patch 5, and hydrogel patch 6).

[0078] FIG. 18 shows BBB scores obtained according to transplantation of a hydrogel patch according to an embodiment (hydrogel patch 2 and hydrogel patch 5).

[0079] FIG. 19 is an image showing histological analysis results obtained according to transplantation of a hydrogel patch according to an embodiment.

MODE OF DISCLOSURE

[0080] Hereinafter, the present disclosure will be described in detail by reference to Reference Examples and Examples. The following Reference Examples and Examples are illustrative of the present disclosure and are not to be construed as limiting the present disclosure.

<EXAMPLES> HYDROGEL BLEND AND PATCH PRODUCTION

[0081] 1-1. Hydrogel Blend

[0082] 20 mg/ml of fibrinogen (Sigma, F8630) was dissolved in DMEM/F12(1:1) (Gibco, 11330-057) culture at a temperature of 37 C. for 30 minutes, 5 mg/ml of a hyaluronic acid (Sigma, 53747) was dissolved in DMEM/F12(1:1) (Gibco, 11330-057) culture at a temperature of 4 C. for a day, and 200 U/ml of thrombin (Sigma, T4648) was dissolved in DMEM/F12(1:1) (Gibco, 11330-057) culture. Collagen (Corning, 354236) was neutralized and ionized in 10 Dulbecco's Phosphate-Buffered Saline (DPBS, Sigma, D5652-10L), deionized water, and 1N NaOH (Millipore, 1.00983.1011), and diluted until the final concentration thereof reached 3.00 mg/mL.

[0083] Then, the above components were blended to prepare a hydrogel in a sol state. The final concentrations of each component are as follows:

[0084] Fibrinogen (Sigma, F8630) 1 mg/ml, 5 mg/mL or 10 mg/ml;

[0085] Laminin (thermofisher, 23017-015) 5 g/ml, 10 g/ml or 50 g/ml;

[0086] Hyaluronic acid (Sigma, 53747) 0.1 mg/ml, 0.5 mg/ml or 1 mg/ml; and

[0087] Collagen (Corning, 354236) 1.2 mg/ml.

[0088] 1-2. Addition of Neuron Growth Factor to Hydrogel Blend

[0089] The hydrogel in a sol state prepared according to Example 1-1 was mixed with the following neuron growth factors and/or vascular endothelial cell growth factors in a culture in which DMEM/F12 (Gibco 11330-057), including penicillin/streptomycin (Invitrogen, 15140-122), 2 mM L-Glutamine (invitrogen, 25030-081), N2 supplement (Gibco, 1750-048), and B27 supplement minus vitamin A (Gibco, 12587010), and neurobasal medium (Gibco, 21103049) were mixed at a ratio of 1:1:

[0090] recombinant Human BDNF (Peprotech, 450-02) 10 ng/ml, recombinant Human GDNF (Peprotech, 450-10) 10 ng/ml, recombinant Human NT-3 (Peprotech, 450-03), 5 ng/ml, db-cAMP (Sigma, D0260) 1 uM, T3 (Sigma, T6397) 60 ng/ml, recombinant Human sonic hedgehog (SHH; Peprotech, 100-45) 50 ng/ml, recombinant Human PDGF-AA (Peprotech, 100-13A-100), recombinant Human FGF-basic (Peprotech, 100-18B) 10 ng/ml, and recombinant Human VEGF165 (Peprotech, 100-20) 20 ng/ml.

[0091] 1-3. Preparation of Hydrogel Patch

[0092] A hydrogel patch was prepared as illustrated in FIG. 1.

[0093] In detail, 5 U/ml of thrombin (Sigma, T4648) was added to the sol-phase hydrogel prepared according to Example 1-1 or 1-2 and incubated at 37 C. for 1 hour for gelation. Then, a parafilm sterilized with ultraviolet light was punched remaining round pores having a size of 0.3 mm to 0.8 mm and the obtained structure was attached on a 10 cm Petri dish. Subsequently, 5 U/ml of thrombin was dispensed into the round pore, and 15 uL to 60 uL of hydrogel was dispensed thereon and the hydrogel and thrombin were mixed while suppressing the formation of bubbles, followed by gelling for about 2 minutes at room temperature, and then subjected to a secondary gelation process at 37 C. for 1 hour. The gel-state hydrogel was separated from the paraffin structure to obtain patches of various sizes ranging from 0.3 mm to 0.8 mm. The prepared hydrogel patch was preserved in the culture including a neuron growth factor and a vascular endothelial growth factor prepared according to Example 1-2.

[0094] The hydrogel prepared by the above method is defined as a hydrogel patch, and types of the hydrogel patch prepared are summarized below.

[0095] In the following description, these hydrogel patches will be referred to as hydrogel patch 1 to 6.

[0096] Hydrogel patch 1: fibrin,

[0097] Hydrogel patch 2: fibrin+laminin+hyaluronic acid

[0098] Hydrogel patch 3: fibrin+laminin+hyaluronic acid+collagen

[0099] Hydrogel patch 4: fibrin+neuron growth factor and vascular endothelial growth factor (all growth factors listed in Example 1-2)

[0100] Hydrogel patch 5: fibrin+laminin+hyaluronic acid+neuron growth factor and vascular endothelial growth factor (all growth factors listed in Example 1-2)

[0101] Hydrogel patch 6: fibrin+laminin+hyaluronic acid+collagen+neuron growth factor and vascular endothelial growth factor (all growth factors listed in Example 1-2)

[0102] 1-4. Formulation of Hydrogel Patch and Possibility for Use of Cryopreservation

[0103] Hydrogel patches were prepared in various formulations, and it was confirmed whether they were able to be cryopreserved.

[0104] In detail, the reversible phase transition of hydrogel patch 2 prepared according to Example 1-3 was confirmed, and the results are shown in FIG. 2.

[0105] As shown in FIG. 2, hydrogel patch 2 was prepared in a liquid formulation in the same manner as in Example 1-1, and immersed in a vial. Thrombin was immersed in a syringe in the same manner as Example 1-3. In addition, hydrogel patch 2 was made into a solid formulation by freeze-drying (Scientific, F78) for 24 hours at 80 C. and 0.33 torr, and then crushed to prepare powder. In addition, a powder formulation was prepared. The above powder formulation was gelled by adding thrombin thereto in the same manner as in Example 1-3, thereby confirming that the powder formulation could return back in the form of gel.

[0106] This result shows that a hydrogel patch according to an embodiment exhibits reversible phase transition, and is able to be provided in various formulations such as solid, semi-solid, liquid or powder formulations.

[0107] In addition, it was confirmed whether cryopreservation was possible.

[0108] In detail, hydrogel patch 2 was added to 10% (v/v) DMSO prepared by adding dimethyl sulfoxide (DMSO, Sigma, D2650) to the culture prepared according to Example 1-2. Thereafter, the temperature was sequentially lowered to 4 C. (30 minutes), 20 C. (1 hour), and 80 C., and the resultant was freeze-dried. The morphological analysis of the hydrogel patch before and after cryopreservation was carried out using a cold FE-SEM (Hitachi High-Technologies, S-4800), and the result is shown in FIG. 3.

[0109] As shown in FIG. 3, there was no morphological difference in the hydrogel patch before cryopreserve and the hydrogel patch that was thawed after one month of cryopreservation. Also, the surface of the hydrogel patch was found to be porous.

<Experimental Example 1> Evaluation of Neural Stem Cell Differentiation In Vitro

[0110] 5-day-old C57BL/6 mice (C57BL/6N Japan SLC, Inc.) were sacrificed and brains thereof were collected, and mouse neural stem cells were primary cell cultured by a known method and cultured in a 100 mm dish (SARSTEDT, 831802).

[0111] A material according to an embodiment and a comparative material were separately applied on the mouse neural stem cells to confirm effects thereof on the differentiation of mouse neural stem cells.

[0112] Mouse neural stem cells were extracted and cultured by using a known method (Kim J B et al. Nat Protoc. 2009), and a hydrogel (fibrin 5 mg/mL; laminin 10 g/ml; and a hyaluronic acid 0.5 mg/ml) prepared according to Example 1-2 was applied on the mouse neural stem cells having the population of 110.sup.4, followed by the addition of thrombin, thereby producing a hydrogel patch. Subsequently, the cells were cultured in a culture containing or not containing the neuron growth factor and vascular endothelial growth factor of Example 1-2. To select the optimal concentration, fibrin alone in an amount of 1 mg/ml, 5 mg/ml, or 10 mg/ml, laminin alone in an amount of 5 g/ml, 10 g/ml, or 50 g/ml, and a hyaluronic acid alone in an amount of 0.1 mg/ml, 0.5 mg/ml, or 1 mg/ml, or laminin in an amount of 10 g/ml, and a hyaluronic acid in an amount of 0.5 mg/ml were used as comparative materials.

[0113] Next, after 14 days of culture, immunofluorescence staining was performed. In detail, for immunocytochemistry, cells were fixed with 4% (w/v) paraformaldehyde in phosphate buffer solution (Wako, 163-20145) for 10 min at room temperature. The fixed cells were diluted 1 with 10 phosphate buffer saline (PBS, P2007), washed three times for 5 minutes at room temperature, and then permeabilized through 0.1% (v/v) triton X-100 (Sigma, T9284) for 10 minutes. After washing three times with 1PBS for 5 minutes at room temperature, the cells were blocked with 4% (v/v) fetal bovine serum dissolved in 1PBS for 1 hour at room temperature to inhibit non-specific binding. Then, the cells were incubated at room temperature for 1 hour by using primary antibody anti-beta III tubulin (Tuj1) (1:400; Abcam) or anti-glial fibrillary acidic protein (GFAP) (1:400; Sigma), and washed three times for 10 minutes at room temperature by using 0.05% (v/v) Tween-20 (Sigma, P7949) (PBST) dissolved three times in 1PBS. Subsequently, light was blocked with secondary fluorescent antibodies (alexa fluorophore-conjugated secondary antibodies 488 (1:1000) or Alexa Fluor594 (1:1000) and then, incubated at room temperature for 30 minutes. When double staining was required, additional blocking was performed at room temperature for 30 minutes before incubation with the other primary antibody. After washing three times for 10 minutes at room temperature with PBST, for cell nuclear staining, the cells were incubated with DAPI (1:1000; Invitrogen) for 15 seconds, and washed three times for 10 minutes at room temperature by using PBST. Cells were stored in PBS for visualization using a fluorescence microscope.

[0114] Subsequently, neuron (Tuj1) and astrocytes (GFAP) were observed by using an inverted fluorescence microscope (Leica, DMI 3000B) with a digital monochrome camera attached (Leica, DFC345 FX). From the image observed with the inverted fluorescence microscope, the number of neurons or astrocytes was counted by using the ImageJ (National Institute of Health (http://rsb.info.nih.gov/ij) software to calculate the differentiation rate.

[0115] All statistical analysis were performed by using unpaired two-tailed Student's t-test. The significance was *P<0.05, **P<0.005, or ***P<0.0005.

[0116] As shown in FIG. 4, when the hydrogel was applied on the neural stem cells according to an embodiment, it was found that the cell junction of the neural stem cells was improved as compared with the bare condition in which the hydrogel was not used.

[0117] As shown in FIG. 5 and FIG. 6, in comparison with fibrin alone, laminin alone, a hyaluronic acid alone, and the combination of laminin and a hyaluronic acid, when the composition according to an embodiment (fibrin, laminin, and a hyaluronic acid) is applied, it can be seen that the differentiation of the neural stem cells into neurons occurred synergistically. In detail, 5% of neurons were present in the control group, and less than 12% of neurons were present in each component alone. However, in the case of the combination of a hyaluronic acid and laminin, about 15% of neurons are present, and when fibrin, which has no substantial effect on its own, was combined with a hyaluronic acid and laminin, it was found that at least 20% of neurons appeared. This suggests that the combination of a hyaluronic acid and laminin is effective in differentiating neurons, and in particular, the combination of the three compositions has a synergistic effect and markedly increases the differentiation into neurons.

[0118] As illustrated in FIG. 7, when the growth factor was added, like the result illustrated in FIG. 6, in comparison with fibrin alone, laminin alone, a hyaluronic acid alone, and the combination of laminin and a hyaluronic acid, when the composition according to an embodiment (fibrin, laminin, and a hyaluronic acid) is applied, it can be seen that the differentiation of the neural stem cells into neurons occurred synergistically. In detail, although the growth factor itself increases the differentiation into neurons, the combination of the three compositions and the growth factor produces a synergistic effect, leading to about 30% of neurons. This result shows that the hydrogel patch or composition according to an embodiment shows a synergistic effect that is unpredictable in general.

[0119] In addition, as illustrated in FIG. 8, each of the growth factors was added to the composition according to an embodiment composition, and as a result, it was confirmed that the neuron differentiation rate of mouse neural stem cells was improved by about 2.5 to 5 times.

[0120] As a result, it can be seen that the composition according to an embodiment of the present disclosure alone produces a synergistic effect of increasing the differentiation rate, and when combined with a growth factor, the combination produces a higher synergistic effect and the effects of growth factor on the cells are increased.

<Experimental Example 2> Evaluation of Biodegradability

[0121] To evaluate biodegradability of the hydrogel patch prepared according to Example 1, 6-week-old C57BL/6 mice (C57BL/6N Japan SLC, Inc.) was anesthetized with 25 mg/ml of Avertin anesthetic, which was prepared by dissolving 2,2,2-tribromoethanol (Sigma, T48402) in tert-amyl alcohol (Sigma, 152463). In this regard, the amount of anesthetic per mouse was 125-250 mg/kg body weight. Subsequently, hydrogel patch 5 was subcutaneously transplanted. Two weeks after the transplantation of the hydrogel patch, it was confirmed whether the subcutaneously transplanted hydrogel patch biodegraded.

[0122] As a result, as illustrated in FIG. 9, it was confirmed that the hydrogel patch according to an embodiment was biodegraded in vivo.

<Experimental Example 3> Spinal Cord Injury (SCI) Treatment Effects of Hydrogel Patch

[0123] 4-1. Confirmation of Treatment Effects of Hydrogel Patch SCI by Using SCI Animal Model

[0124] To confirm SCI regeneration effects of a hydrogel patch according to an embodiment, a gel patch was transplanted into a SCI animal model and then a behavioral test was performed thereon for 8 weeks.

[0125] An evaluation method of official small SCI animal model is to evaluate behavioral analysis of chronic SCI study, in which joints, hind legs, gait, harmony movement of forelegs and hind legs, the position of waist, support by soles, and the position of tails are observed and evaluated to identify the recovery of the behavioral ability. The obtained evaluation result of each animal, that is, the recovery ability was quantified at a scale of 0 to 21: the scale of 0 to 7 indicates an early stage in which the minimal behavioral ability was recovered, that is, the movement of the hind legs were hardly recovered, and the scale of 8 to 13 indicates an intermediate recovery stage in which the movement of the hind legs was recovered, but there was a gap in the harmonic motion in which the forelegs and hind legs were not well controlled. Finally, the scale of 14 to 21 indicates a late recovery stage in which forelegs and hind legs move harmonically.

[0126] First, adult male Sprague-Dawley rats (OrientBio) was anesthetized by injection of 10 mg/rat Zoletil 50 (Virbac), and then, the spinal cord lamina of T9 site was removed therefrom. Then, the spinal cord was compressed for 10 minutes by using a vascular clip, thereby completing the preparation of a SCI animal model. After the SCI surgery, the bladder was compressed twice a day for about 2 weeks until the rates had voluntary urination. One week after the preparation of the SCI animal model, as shown in FIG. 10, a hydrogel patch according to an embodiment was directly transplanted in the SCI site. Behavioral evaluation was performed by using the SCI animal model evaluation method (BBB test; open field test) for 8 weeks, and the average of observation results of two observers the test results was used as test results, and the results are shown in FIGS. 15 to 18.

[0127] FIG. 11 shows an image of a SCI animal model to observe the movement of hind legs thereof, one week after the SCI. FIG. 12 shows an image of a SCI animal model to observe the movement of hind legs thereof, eight weeks after the SCI, when PBS alone, without the hydrogel patch, is applied on the transplantation site. FIG. 13 shows an image of a SCI animal model to observe the movement of hind legs thereof, eight weeks after the SCI, when hydrogel patch 2 (hydrogel patch 2 without GF) is transplanted. FIG. 14 shows an image of a SCI animal model to observe the movement of hind legs thereof, eight weeks after the SCI, when hydrogel patch 5 (hydrogel patch 2 with GF) is transplanted.

[0128] FIGS. 15 to 18 show BBB scores obtained according to transplantation of a hydrogel patch according to an embodiment.

[0129] As shown in FIGS. 11 to 14, it was confirmed that when PBS alone was applied on the transplantation site without the transplantation of the hydrogel patch, the SCI animal model showed no change in the motor ability of hind legs 8 weeks after the SCI. However, when hydrogel patch 2 was transplanted, the SCI animal model recovered its motor ability of a left hind leg 8 weeks after SCI, and when hydrogel patch 5 including a neuron growth factor and a vascular endothelial growth factor was planted, the SCI animal model remarkably recovered its motor ability of both hind legs 8 weeks after SCI.

[0130] In addition, the SCI recovery ability was evaluated by using a SCI animal model evaluation method (BBB test; open field test). The evaluation results show that, as illustrated in FIGS. 15 to 18, when PBS alone was applied on the transplantation site without the transplantation of the hydrogel patch, the SCI animal model had the BBB score of 4 to 6. This belongs to the early stage of the SCI regeneration process. However, when hydrogel patch 2 was transplanted, the SCI animal model had the BBB score of about 8 to about 10. This indicates an intermediate recovery stage of the SCI regeneration process. When hydrogel patch 5 was transplanted, the SCI animal model had the BBB score of about 10 to about 12. This indicates the late recovery state of the SCI regeneration process.

[0131] As illustrated in FIGS. 15 to 18, when hydrogel patch 1 or hydrogel patch 4 were transplanted, the SCI animal model had the BBB score of about 6 to about 8. This indicates the early stage of the SCI regeneration process. These results indicate that hydrogel patches containing fibrin, laminin, and a hyaluronic acid have a remarkable effect compared to other combinations.

[0132] In addition, when hydrogel patch 2 and hydrogel patch 5 were compared with hydrogel patch 3 and hydrogel patch 6 which include collagen, it was confirmed that there is no significant difference in the BBB scores.

[0133] In addition, hydrogel patch 3 was compared with the combination of hydrogel patch 3 and PBS, and it was found that there is no significant difference in the BBB scores. This result shows that a cell culture does not affect the efficacy of the hydrogel patch.

[0134] Therefore, it was confirmed that a hydrogel patch according to an embodiment has a therapeutic effect of remarkably recovering the motor ability lost by SCI.

[0135] 4-2. Confirmation of Treatment Effects of Hydrogel Patch SCI by Using Histological Analysis

[0136] The SCI regeneration effect of a hydrogel patch according to an embodiment was confirmed by histological analysis.

[0137] In detail, for histological analysis to identify the neuron regeneration effects of a hydrogel patch on the SCI site, rats were scarif iced 8 weeks after the hydrogel patch transplantation surgery, and then, perfused by using 4% (w/v) paraformaldehyde (Merck) to collect a spinal cord. Thereafter, the spinal cord was cut to a thickness of 10 m, and the cut spinal cord sample was stained in the same manner as in Experimental Example 1 by using anti-neurofilament (NF) (1:3000, Abcam), anti-GFAP (1:1000, Abcam), anti-2,3-Cyclic-nucleotide 3-phosphodiesterase (CNPase) (1:800, Abcam), anti-homeobox HB9 (Hb9) (1:100, DSHB), and anti-ionized calcium-binding adapter molecule 1 (lba-1) (1:500,abcam). Thereafter, the hydrogel transplanted site was photographed by using a cofocal microscope (LSM 700, Zeiss) to evaluate the nerve regeneration. The results thereof are shown in FIG. 19.

[0138] As a result, as illustrated in FIG. 19, compared to when PBS was applied on the transplantation site, when hydrogel patch 2 or hydrogel patch 5 was transplanted, in SCI lesions, the distribution of GFAP astrocytes was low while the distribution of NF neurons was high. In addition, when the hydrogel patch 2 or hydrogel patch 5 was transplanted, the CNPase oligodendrocyte was co-localized at the same position as the NF neuron. These results show that due to the applying with hydrogel patch, the regeneration of injured neurons and the formation of myelin sheath of neurons were significantly increased.

[0139] Also, when PBS was applied on the transplantation site, the distribution of NF neurons was small and the distribution of lba-1 microglial cells, which show inflammatory response, was high. However, when hydrogel patch 2 or hydrogel patch 5 was transplanted, the distribution of motor neurons, which express Hb9 and NF of the spinal cord, was high, and the distribution of lba-1 microglial cells was substantially decreased. These results indicate that inflammatory responses, caused by the SCI when the hydrogel patch is transplanted, was reduced and thus the nerve injury was inhibited, and the regeneration of motor neurons and oligodendrocyte was promoted and the regeneration of the motor neuron with myelin sheath was promoted.

[0140] These results indicate that the hydrogel patch has substantially increased therapeutic effects of inducing the regeneration of injured spinal cord and inhibiting the activation of astrocytes to recover the lost motor ability, and the secondary SCI, caused by the SCI treatment using a syringe, may be substantially inhibited by using the hydrogel patch.