WO2018169314A1

WO2018169314A1 - Hydrogel patch

Info

Publication number: WO2018169314A1
Application number: PCT/KR2018/003011
Authority: WO
Inventors: 김정범
Original assignee: 울산과학기술원
Priority date: 2017-03-14
Filing date: 2018-03-14
Publication date: 2018-09-20

Abstract

The present invention relates to a hydrogel patch, a preparation method therefor, and a composition comprising the same for treatment of spinal cord injury. A hydrogel patch according to one embodiment has the effect of being able to treat a spinal cord injury patient in a non-invasive manner for the spinal cord.

Description

Hydrogel patch

The present invention relates to a hydrogel patch, a method for preparing the same, and a composition for treating spinal cord injury, including the same.

Damage to the central nervous system (spinal spinal cord) involves psychological as well as medical disorders of motor and urinary disorders. Currently, in order to alleviate nerve damage with spinal cord injury treatment, surgical treatment, steroid-based drug treatment such as methylprednisolone, or rehabilitation are being performed. However, this is only a treatment to alleviate the impairment caused by nerve damage, and there is no fundamental spinal nerve regeneration treatment for impaired spinal nerve regeneration.

How to create a biological nerve regeneration induction environment in the nerve injury site to induce damaged spinal nerve regeneration, or cell therapy to administer neural stem cells, oligodendrocyte progenitor cells, mesenchymal stem cells, Schwanse cells or olfactory nerve shell cells Is being developed. Adult stem cells, embryonic stem cells, or induced pluripotent stem cells are mainly used for the development of cell therapies for use in cell therapy.

However, in adult stem cells, there are problems of scarcity, limited differentiation capacity, and immune rejection reactions due to the use of multivalent cells. In addition, in the case of embryonic stem cells, there is a problem of ethical problems and immune rejection reactions due to the use of multivalent cells, and embryonic stem cells and induced pluripotent stem cells commonly have problems of carcinogenicity.

In spinal cord injury, 48 hours after injury, which is an acute period of spinal cord injury, is known as a titration for spinal cord injury treatment. Cell therapy requires neural regenerative cells such as autologous neural stem cells and oligodendrocyte progenitor cells in order to evade immune rejection.However, by collecting the cells from patients with acute spinal cord spinal cord injury, securing the number of cells to be used as cell therapy within an acute period. Is an impossible reality. In addition, induced pluripotent stem cells or direct cross-differentiated cells produced from patient somatic cells can satisfy the number of cells required for the preparation of cell therapy products by self-renewal, but the time to produce them and the period of differentiation into target cells are not suitable for acute treatment. Treatment is not possible in the acute period, which is the optimal time for spinal cord injury treatment.

Therefore, there is a need to develop a fundamental spinal nerve regeneration technology that minimizes damage to the spinal cord and induces regeneration of the damaged spinal cord by non-invasive spinal cord nerves in a timely manner.

One aspect includes fibrin and / or fibrinogen; Laminin or laminin derived peptides or proteins; And / or a hydrogel patch comprising hyaluronic acid or a salt thereof.

Other aspects include peptides derived from fibrinogen, laminin or laminin; And / or adding thrombin to a composition in a sol (Sol) state comprising hyaluronic acid or a salt thereof.

Another aspect is fibrin and / or fibrinogen; Laminin or laminin derived peptides or proteins; And / or hyaluronic acid or a salt thereof.

Another aspect is a laminin or laminin derived peptide or protein; And hyaluronic acid or a salt thereof.

Another aspect is to provide a method of cryopreserving or cryopreserving the hydrogel patch at 4 to -210 ° C in solution.

One aspect provides a hydrogel patch.

Another aspect provides a pharmaceutical composition.

Another aspect provides a method of treating or preventing a disease, eg, spinal cord injury, comprising administering the hydrogel patch or composition to a subject.

Another aspect provides the use of the hydrogel patch or composition for the manufacture of a therapeutic agent for a disease.

The hydrogel patch or composition may comprise fibrin and / or fibrinogen; Laminin or laminin derived peptides or proteins; And / or hyaluronic acid or salts thereof. For example, the hydrogel patch or composition may be a laminin or laminin derived peptide or protein; And hyaluronic acid or salts thereof.

The term "treatment" refers to or includes the alleviation, inhibition or prevention of a disease, disorder or condition, or one or more symptoms thereof, and a "pharmaceutically effective amount" means alleviation of a disease, disorder or condition, or one or more symptoms thereof. It can mean any amount of the composition used in the practice of the invention provided herein, sufficient to inhibit or prevent progression.

The terms “administering,” “applying,” “introducing,” and “transplanting” are used interchangeably and in a method or route that results in at least partial localization of a patch or composition to a desired site in accordance with one embodiment. By means of placement of a patch or composition according to one embodiment into an individual.

As used herein, the term "patch" can mean a means that has a certain shape and that can be applied, attached, or contacted to a desired site.

In one embodiment, the hydrogel patch or composition can be one that has an intermediate property between a solid and a liquid. The hydrogel patch or composition may be amorphous, spherical, hemispherical, discotic, or cylindrical. Also, for example, the diameter of the hydrogel patch may be 0.05 mm to 10 cm, 0.1 mm to 5 cm, 0.1 mm to 3 cm, or 0.2 mm to 1.5 cm, and may be provided in such size or shape. . In addition, the hydrogel patch may be modified in conformity with the shape of the injury site by applying, implanting, attaching, or contacting the target site (for example, tissue damage site).

In another embodiment, the hydrogel patch or composition may be solid (including powder), semisolid, or liquid. In addition, for example, the hydrogel patch or composition may undergo a reversible phase transition (eg, with temperature changes) to a solid (including powder), semisolid, or liquid state. Since the hydrogel patch may undergo a reversible phase transition according to surrounding conditions such as temperature conditions, the hydrogel patch according to one embodiment may be produced or provided in a solid (including powder) or liquid state, before administration to a target site, At the time of administration or after administration, it can be converted into a hydrogel patch and used. For example, the hydrogel patch may be provided in a sol state containing fibrinogen, laminin, and hyaluronic acid, so that a user can convert fibrinogen into fibrin (eg, thrombin), Hydrogel patch according to one embodiment can be prepared and used. Thus, hydrogel patches according to one embodiment may include fibrin and / or fibrinogen; Laminin; And / or compositions comprising hyaluronic acid, for example, in the form of prodrugs of solid (powder), liquid (sol), or semisolid compositions. Compositions provided in the form of prodrugs may be prepared, or modified, in a hydrogel patch in vivo. In addition, the hydrogel patch according to one embodiment may further comprise thrombin, or thrombin may be provided together as a kit.

In yet another embodiment, the hydrogel patch or composition can be porous. In detail, the surface of the hydrogel patch may have porosity (micropores). Without being limited to a particular theory, the hydrogel patch according to one embodiment has a porosity, thereby enhancing interaction between the active materials.

The hydrogel patch or composition may be used in a non-invasive manner of spinal cord in the spinal cord injury site of the patient. The non-invasive method herein is distinguished from the cell therapy or drug administration of the invasive method in the spinal cord using a needle, the hydrogel patch or composition is implanted in the target site without other means of delivery (for example, syringe) Can be applied, attached or contacted. The non-invasive spinal cord can be used in a non-invasive manner by injecting a therapeutic composition into the spinal cord, such as a needle, to cause spinal cord injury at the injection site due to the needle. It may mean a method of delivering the composition to the injury site without spinal cord injury caused by the injection method of the composition through the injection needle through the application or implantation to the injury site. In addition, the hydrogel batch or composition may serve to prevent secondary damage of the spinal cord caused by the invasive method. In addition, the hydrogel patch may be biodegraded in vivo after a certain time.

In one embodiment, the hydrogel patch or composition may comprise fibrin and / or fibrinogen. Final pharmacological agents acting in vivo include fibrin, but fibrinogen may be used in place of fibrin in the form of prodrugs. Further, depending on the amount of the substance converting fibrin to fibrinogen, the hydrogel patch or composition according to one embodiment may include some fibrinogen or thrombin. Thus, the present specification relates to fibrinogen; Laminin; And / or prodrugs comprising hyaluronic acid. The fibrin or fibrinogen is at a concentration of 0.5 to 20 mg / ml, 0.8 to 16 mg / ml, 0.8 to 12 mg / ml, 1.0 to 10 mg / ml, 2 to 8 mg / ml, or 4 to 8 mg / ml. May be included.

The fibrinogen glycoprotein is a hexamer consisting of soluble α, β, and γ subunits made in liver cells of the liver. Fibrinogen reacts with a thrombin enzyme to phase change into a soluble to insoluble fibrin polymer fiber.

The thrombin enzyme is a serine protease and is an enzyme that transforms soluble fibrinogen into insoluble fibrin. The thrombin may act to gel the hydrogel in sol state by converting fibrinogen into fibrin.

As used herein, the term "laminin" refers to a heterotrimeric protein consisting of α, β, and γ subunits as an extracellular matrix protein constituting a basal lamina. Thus, the laminin may comprise laminin-derived peptides or proteins as well as laminin full-length proteins. For example, the laminin may be laminin-1, laminin-2, laminin-3, laminin-4, laminin-5A, laminin-5B, laminin-6, laminin-7, laminin-8, laminin-9, laminin-10. , Laminin-11, laminin-12, laminin-14, or laminin-15. In addition, the laminin-derived peptide may be α chain, γ chain, or β chain. The laminin may be included at a concentration of 1 to 100 μg / ml, 2 to 80 μg / ml, 5 to 50 μg / ml, 5 to 25 μg / ml, 8 to 20 μg / ml, or 8 to 15 μg / ml. have.

The hyaluronic acid (glycosaminoglycan) is a glycoside bond having a change of β- (1 → 4) and β- (1 → 3) between D-glucuronic acid and N-acetyl-D-glucosamine. It is a polysaccharide of disaccharide bond consisting of glycosidic bonds) and has various molecular weights depending on the length of disaccharide bond. In one embodiment, the molecular weight of hyaluronic acid may be 5,000 to 20,000,000 Da. More specifically, the molecular weight of hyaluronic acid may be 0.5 to 4.0 x 10 ⁶ Da, 1.0 to 2.0 x 10 ⁶ Da, or 1.5 to 1.8 x 10 ⁶ Da. The hyaluronic acid is 10 μg / ml to 5 mg / ml, 50 μg / ml to 5 mg / ml, 100 μg / ml to 3 mg / ml, 100 μg / ml to 1 mg / ml, 200 μg / ml to 1 mg / ml, or at a concentration of 300 μg / ml to 800 μg / ml. In addition, the hyaluronic acid may be provided in the form of a salt, a pharmaceutically acceptable salt, for example, sodium salt, potassium salt, calcium salt, magnesium salt and the like.

In another embodiment, the hydrogel patch or composition may further comprise a cell growth factor. The cell growth factor, neuronal growth factor, vascular endothelial growth factor, fibroblast growth factor, bone morphogenetic protein, epidermal growth factor, hepatocyte growth factor, transforming growth factor or their May be a combination. More specifically, the growth factor is placental growth factor, macrophage colony stimulating factor, granulocyte macrophage colony stimulating factor, neurophylline, fibroblast growth factor (FGF) -1, FGF-2 (bFGF), FGF-3, FGF -4, FGF-5, FGF-6, erythropoietin, BMP-2, BMP-4, BMP-7, TGF-beta, IGF-1, Osteopontin, Piotropin, Activin, Endo Mullin-1 and combinations thereof. The neuronal growth factor may be a brain-derived neurotropic factor (BDNF), a cellular cell derived neurotropic factor (GDNF), a ciliary neurotropic factor (CNTF), a basic fibroblast growth factor (bFGF), a cyclic adenocyne monophosphate (cAMP), or a neurotropin (NT). , At least one selected from the group consisting of NT3 (Neurotropin-3), NT4 (Neurotropin-4), T3 (Triiodo-L-Thyronine), SHH (Sonic hedgehog), and PDGF (Platelet-derived growth factor) It may be. The vascular endothelial growth factor may include vascular endothelial growth factor (VEGF) -A, VEGF-A, VEGF-B, VEGF-C, VEGF-D, or VEGF-E. The cell growth factor may be different from the concentration contained in the hydrogel patch or composition according to the type of growth factor, but may generally be included in a concentration of 1 ng / ml to 1,000 ng / ml or 0.1 μM to 100 μM. The cell growth factor may play a role in enhancing the tissue damage repair effect of the hydrogel patch or composition comprising fibrin, laminin, and hyaluronic acid.

The brain derived neurotrophic factor (BDNF) protein is encoded by the BDNF gene. BDNF proteins are known to assist in the survival of central nervous system neurons and the differentiation and growth of new neurons.

The Glial cell line-derived neurotrophic factor (GDNF) protein is encoded by the GDNF gene. GDNF protein is known to help the survival and differentiation of dopaminergic neurons and motor neurons of the central nervous system.

The Ciliary neurotrophic factor (CNTF) protein is encoded by the CNTF gene. CNTF proteins promote the production of neurotransmitters and are known to help neurons and oligodendrocytes survive.

The Neurotrophin-3 (NT-3) protein is encoded by the NT-3 gene. NT-3 protein is known to assist in the survival of central nervous system neurons and the differentiation and growth of new neurons.

Cyclic adenosine monophosphate (cAMP) protein is a protein derived from adenosine triphosphate (ATP) under the action of adenylate cyclase, and serves as a second messenger of cells. Neurons are known to be involved in the control of neurotransmitters, such as the production, storage, and distribution of neurotransmitters. They are known to help neuronal survival and differentiation. It is known to play a role in the production of nitric oxide.

The Sonic Hedgehog (SHH) protein is encoded by the SHH gene. SHH plays a role in hedgehog signaling and is known to play a role in the differentiation of motor neurons, particularly in the nervous system.

The triiodothyronine (T3) hormone (Hormone) is a type of thyroid hormone and affects most physiological roles in vivo. In the nervous system, in particular, it is known to play a role in inhibiting proliferation of oligodendrocyte progenitor cells and promoting differentiation into oligodendrocytes.

Subtypes of the platelet-derived growth factor (PDGF) include -AA, -BB, -AB, -CC, -DD and PDGF- in the homodimer of the PDGFA subunit encoded from the Platelet derived growth factor subunit A (PDGFA) gene. AA and each subtype has a different PDGF receptor (PDGFR). Platelet-derived growth factor-AA (PDGF-AA) is a dimeric glycoprotein, especially in the nervous system, along with Fibroblast growth factor (FGF), which activates the PDGF receptor of oligodendrocyte progenitor cells. It is known to play a role in maintaining proliferation.

The Vascular endothelial growth factor (VEGF) protein is a type of Vascular Permeability Factor (VPF) that plays a role in angiogenesis and vascularization. In particular, VEGF is known to play a role in the formation of new blood vessels such as embryonic development, wound, etc., and is known to help nerve cell survival by preventing neuronal death due to toxicity and stress.

In other embodiments, the hydrogel patch or composition may or may not comprise substantially collagen. Without being limited to a particular theory, the collagen may or may not be substantially included as a component of the composition, but may be more advantageous in some aspects than what is not included.

In addition, the hydrogel patch or composition may be substantially free of cells. As used herein, "substantially does not comprise" means that collagen or cells are included or not included to the extent that they will not affect the activity, or pharmacological activity, of the hydrogel patch or composition. The hydrogel patch or composition according to one embodiment does not substantially include cells, and thus may be distinguished from a cell therapy agent generally used for regeneration of damaged tissue.

The collagen (Collagen) protein is typically divided into Type I, II, III, IV, V and Type I collagen is most present in the body. Collagen is a triple helix structure of α1 and α2.

In one embodiment, the hydrogel patch or composition comprises fibrin and / or fibrinogen; Laminin; And hyaluronic acid or salts thereof. In addition to this, one or more of the components mentioned herein may be additionally included. For example, the hydrogel patch or composition may comprise fibrin and / or fibrinogen; Laminin; Hyaluronic acid or a salt thereof; Optionally thrombin; Optionally collagen; And optionally cell growth factors.

In one embodiment, the hydrogel patch or composition may be to enhance cell adhesion. Thus, the present disclosure additionally provides a method of enhancing adhesion of cells (eg neurons) of an individual comprising administering the hydrogel patch or composition to the individual.

In another embodiment, the hydrogel patch or composition may be to increase the differentiation of neural stem cells into neurons. Accordingly, the present disclosure further provides a method of increasing the differentiation of neural stem cells into neurons of an individual comprising administering the hydrogel patch or composition to the individual.

In another embodiment, the hydrogel patch or composition may be to reduce the inflammatory response to inhibit nerve damage, or to improve the regeneration of motor nerve cells formed myelin by helping regeneration of motor neurons and oligodendrocytes. .

In another embodiment, the hydrogel patch or composition may function to induce nerve regeneration damaged by neurodegeneration, such as nerves and strokes damaged by trauma such as spinal cord injury. Accordingly, the present disclosure further provides a method of inhibiting damage to nerve cells, inducing nerve regeneration, or regenerating motor neurons in a subject, comprising administering the hydrogel patch or composition to the subject.

In another embodiment, the hydrogel patch or composition may be to inhibit spinal cord secondary damage caused by spinal cord injury treatment.

Thus, the hydrogel patch or composition according to one embodiment may be for regeneration or repair of damaged tissue. Specifically, the damaged tissue may be the spinal cord.

Spinal cord injury (SCI) herein may mean damage due to spinal cord compression, dislocation. Spinal cord injuries include traumatic spinal cord injury, spinal degenerative diseases (such as spondylosis), spinal inflammatory diseases (spinalitis, chronic rheumatoid arthritis, etc.), tumors (spinal cord tumors, spinal tumors, etc.), and vascular diseases (spinal bleeding, stroke, extramedullary vascular disorders). Spinal cord paralysis, etc.), myelitis (spider meningitis, viral myelitis, bacterial myelitis, etc.), multiple sclerosis, amyotrophic lateral sclerosis, and the like. Hydrogel patch or composition according to one embodiment may have a therapeutic effect in addition to acute spinal cord injury, as well as chronic spinal cord injury. Thus, the spinal cord injury may be chronic spinal cord injury.

The dosage of the composition according to one embodiment may be 0.01 mg to 10,000 mg, 0.1 mg to 1000 mg, 1 mg to 100 mg, 0.01 mg to 1000 mg, 0.01 mg to 100 mg, 0.01 mg to 10 mg, or 0.01 mg to 1 mg. However, the dosage may be variously prescribed by such factors as the formulation method, the mode of administration, the age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion, and reaction sensitivity of the patient. These factors can be taken into account to properly adjust the dosage. The number of administrations may be one or two or more times within the range of clinically acceptable side effects, and may be administered to one or two or more sites of administration. In the case of animals other than humans, the same dosages as humans per kg or the above dosages are converted into volume ratios (for example, average values) of organs (heart, etc.) between the target animal and humans. One dose may be administered. Possible routes of administration include oral, sublingual, parenteral (eg, subcutaneous, intramuscular, intraarterial, intraperitoneal, intradural, or intravenous), rectal, topical (including transdermal), inhalation, and injection, or implantable devices Or insertion of material. Examples of the target animal for the treatment according to one embodiment include humans and mammals for other purposes, and specifically, humans, monkeys, mice, rats, rabbits, sheep, cattle, dogs, horses, pigs, and the like. Included.

Pharmaceutical compositions according to one embodiment may comprise a pharmaceutically acceptable carrier and / or additive. Examples include sterile water, physiological saline, conventional buffers (phosphate, citric acid, other organic acids, etc.), stabilizers, salts, antioxidants (ascorbic acid, etc.), surfactants, suspending agents, isotonic agents, or preservatives. can do. For topical administration, this may include combining organic materials such as biopolymers, inorganic materials such as hydroxyapatite, specifically collagen matrix, polylactic acid polymers or copolymers, polyethylene glycol polymers or copolymers, and chemical derivatives thereof. Can be.

The pharmaceutical composition according to one embodiment may be a suspension, dissolution aid, stabilizer, tonicity agent, preservative, adsorption agent, surfactant, diluent, excipient, pH adjuster, painless agent, Buffers, reducing agents, antioxidants and the like may be included as appropriate. Pharmaceutically acceptable carriers and formulations suitable for the present invention, including those exemplified above, are described in detail in Remington's Pharmaceutical Sciences, 19th ed., 1995. A pharmaceutical composition according to one embodiment is in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient, according to methods which may be readily implemented by one of ordinary skill in the art. It can be prepared by or incorporated into a multi-dose container. The formulations can then be in the form of solutions, suspensions or emulsions in oil or aqueous media or in the form of powders, granules, tablets or capsules.

Another aspect is fibrinogen; Laminin or laminin derived peptides; And thrombin added to a composition in a sol (Sol) state comprising hyaluronic acid or a pharmaceutically acceptable salt thereof.

The method may further comprise formulating a cell growth factor in the sol state composition.

The method may also include gelling with thrombin followed by secondary gelling with thrombin again. The gelation may be performed at 10 to 40 ℃ for 5 minutes to 3 hours. In addition, the thrombin may be added at a concentration of about 1 to 10 U / ml. The hydrogel patch may be manufactured in various shapes or sizes depending on the shape of the mold.

The method may also include cryopreserving or cryopreserving the hydrogel patch at 4 to -210 ° C in solution. The solution may include DMSO (dimethyl sulfoxide), but any solution may be used as long as it does not substantially change the chemical or physical properties of the hydrogel patch. The hydrogel patch does not substantially change its form or activity even when cryopreserved or cryopreserved.

The hydrogel patch, fibrin and / or fibrinogen, laminin, hyaluronic acid or cell growth factors are as described above.

Another aspect provides a method of cryopreserving or cryopreserving the hydrogel patch at 4 to -210 ° C in solution (eg DMSO).

The hydrogel patch, cryopreserved or cryopreserved by the above method, may be used immediately when a patient develops to treat acute stage spinal cord injuries, which is a proper stage for spinal cord injury treatment, and even if it is melted after being transferred to room temperature from cryopreservation or cryopreservation There may be no change and no significant difference in the effects of spinal cord injury treatment.

The present invention relates to a hydrogel patch and a method of manufacturing the hydrogel patch. According to the hydrogel patch according to one aspect, there is an effect that can be used for various diseases including spinal cord injury by repairing damaged tissue.

1 is a view showing a hydrogel patch manufacturing method and a hydrogel patch of various sizes of 0.3 mm to 0.8 mm according to an embodiment.

FIG. 2 shows a hydrogel patch formulation (liquid, gel, powder) (top panel, left to right) and gelation of the powder formulation (bottom panel) according to one embodiment.

FIG. 3 is an image showing before and after cryopreservation of a cross section of the hydrogel patch according to one embodiment, the outer surface of which is observed by scanning electron scanning microscope (SEM).

Figure 4 shows the effects of in vitro mouse neural stem cell differentiation of the composition of the hydrogel patch (fibrin, hyaluronic acid, and laminin) and the bare group according to an embodiment on light microscopy and immunity. Figure confirmed using fluorescence staining; Scale bars represent 100 μm and 50 μm, respectively.

Figure 5 shows the effect on the in vitro mouse neural stem cell differentiation according to each component of the hydrogel patch and combination of the two components according to one embodiment using neurofluorescence staining (Tuj1) , Astrocytes (GFAP) differentiation distribution image; Scale bars represent 50 μm.

Figure 6 quantifies the effect on the in vitro mouse neural stem cell differentiation according to the hydrogel patch, each component alone, and the combination of the two components according to one embodiment in a neuronal cell (Tuj1) differentiation distribution It is a graph shown; * p <0.05, ** p <0.005, *** p <0.0005.

FIG. 7 shows the effect of neuronal cells on the in vitro mouse neural stem cell differentiation by adding growth factors to the hydrogel patch, its components alone, and a combination of the two components according to one embodiment. Tuj1) is a graph quantified by differentiation distribution; * p <0.05, ** p <0.005, *** p <0.0005.

FIG. 8 shows each growth factor (FGF, BDNF, GDNF, cAMP, NT3, PDGF-AA, SHH, T3, or VEGF) added to a hydrogel patch according to one embodiment thereof, in vitro mice Influence on neural stem cell differentiation is a graph quantifying the neuronal cell (Tuj1) differentiation distribution; * p <0.05, ** p <0.005, *** p <0.0005.

9 is an image showing biodegradation when the hydrogel patch is implanted in vivo according to an embodiment.

FIG. 10 is an image illustrating a process of implanting a hydrogel patch into an injured spinal cord according to one embodiment. FIG.

11 is a view of the hind limb movement of the spinal cord injury animal model lost to spinal cord injury after one week of spinal cord injury animal model.

12 is a view of the hind limb movement of the spinal cord injury week 8 of the spinal cord injury animal model in which only PBS was applied to the implantation site without implantation of the hydrogel patch.

FIG. 13 is a diagram illustrating hind limb movement at 8 weeks of spinal cord injury in a spinal cord injury animal model implanted with Hydrogel patch 2 without GF.

FIG. 14 is a diagram illustrating hind limb movement at week 8 of spinal cord injury in a spinal cord injury animal model implanted with Hydrogel patch 5 (GF).

FIG. 15 is a graph showing BBB score values according to hydrogel patch (Patch 3 with PBS, Patch 3 and Patch 6) transplants according to one embodiment. FIG.

FIG. 16 is a graph showing BBB score values following implantation of hydrogel patches (Patch 1, Patch 2, and Patch 3) according to one embodiment. FIG.

FIG. 17 is a graph showing BBB score values following implantation of hydrogel patches (Patch 4, Patch 5, and Patch 6) according to one embodiment. FIG.

FIG. 18 is a graph showing BBB score values following hydrogel patch (patch 2, and patch 5) implantation according to one embodiment. FIG.

19 is an image showing the results of histological analysis following the implantation of a hydrogel patch according to one embodiment.

Hereinafter, the present invention will be described in detail by reference examples and examples. However, the following reference examples and examples are merely to illustrate the invention and should not be construed as limiting the invention.

<실시예> 하이드로젤 배합 및 패치 제작<Example> Hydrogel formulation and patch production

1-1. 하이드로젤 배합1-1. Hydrogel formulation

Dissolve 30 minutes of fibrinogen (Sigma, F8630) at 37 ° C. in DMEM / F12 (1: 1) (Gibco, 11330-057) medium at 20 ° C / ml, and DMEM / F12 (1: 1) (Gibco, 11330 -057) Dissolve hyaluronic acid (Sigma, 53747) in culture at 5 ° C. for 1 day at 4 ° C., and thrombin (Sigma, T4648) in DMEM / F12 (1: 1) (Gibco, 11330-057) culture. It melt | dissolved so that it might become 200 U / ml. Collagen (Corning, 354236) was neutralized and ionized in 10X Dulbecco's Phosphate-Buffered Saline (DPBS, Sigma, D5652-10L), 3rd order, and 1N NaOH (Millipore, 1.00983.1011) to give a final concentration of 3.00 mg / mL. Dilute as much as possible.

Thereafter, the above ingredients were combined to prepare a hydrogel in sol state. The final concentration of each component is as follows:

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

Laminin (Thermofisher, 23017-015) 5 μg / ml, 10 μg / ml or 50 μg / ml;

Hyaluronic acid (Sigma, 53747) 0.1 mg / ml, 0.5 mg / ml or 1 mg / ml; And

Collagen (Corning, 354236) 1.2 mg / ml.

1-2. 하이드로젤 배합에 신경세포 성장인자의 첨가1-2. Addition of Neuronal Growth Factors to Hydrogel Formulations

Penicillin / Streptomycin (Invitrogen, 15140-122), 2 mM L-Glutamine (invitrogen, 25030-081), N2 supplement (Gibco, 1750-048) for the sol hydrogel formulated in Example 1-1, and Neuronal growth factors and / or vascular endothelial cells in a 1: 1 mixture of DMEM / F12 (Gibco 11330-057) and Neurobasal medium (Gibco, 21103049) containing B27 supplement minus vitamin A (Gibco, 12587010) Add growth factor was combined:

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.

1-3. 하이드로젤 패치의 제작1-3. Fabrication of Hydrogel Patches

As shown schematically in FIG. 1, a hydrogel patch was prepared.

Specifically, 5 U / ml thrombin (Sigma, T4648) was added to the sol hydrogel formulated in Example 1-1 or 1-2, incubated at 37 ° C. for 1 hour, and then gelled. The original sterilized parafilm was taken in a circular frame of 0.3 to 0.8 mm and attached to 10 cm petridish. Subsequently, after dispensing 5 U / ml of thrombin in a circle, dispense 15 to 60 uL of hydrogel and mix hydrogel and thrombin so that no bubbles are generated in the hydrogel, followed by gelation at room temperature for about 2 minutes. Secondary gelation was performed at 37 ° C. for 1 hour. The hydrogel in the gel state was separated from the parafilm of the frame and manufactured in the form of patches of various sizes from 0.3 mm to 0.8 mm. The prepared hydrogel patch was preserved in the culture medium to which the neuronal growth factor and vascular endothelial growth factor of Example 1-2 were added.

The hydrogel form produced by the above method is called a hydrogel patch, and the types of hydrogel patches produced are summarized below.

Hereinafter, the hydrogel patches 1 to 6 will be described.

Hydrogel patch 1: fibrin,

Hydrogel Patch 2: Fibrin + Laminin + Hyaluronic Acid

Hydrogel Patch 3: Fibrin + Laminin + Hyaluronic Acid + Collagen

Hydrogel Patch 4: Fibrin + Neuronal Growth Factor and Vascular Endothelial Growth Factor (all growth factors described in Examples 1-2)

Hydrogel Patch 5: Fibrin + Laminin + Hyaluronic Acid + Neuronal Growth Factor and Vascular Endothelial Growth Factor (all growth factors described in Examples 1-2)

Hydrogel Patch 6: Fibrin + Laminin + Hyaluronic Acid + Collagen + Neuronal Growth Factor and Vascular Endothelial Growth Factor (all growth factors described in Examples 1-2)

1-4. 하이드로젤 패치의 제형 및 동결보존 가능 여부 확인 1-4. Formulation of Hydrogel Patches and Confirmation of Cryopreservation

Various formulations of the prepared hydrogel patches were prepared and their cryopreservation was confirmed.

Specifically, the reversible phase transition of the hydrogel patch 2 prepared in the same manner as in Example 1-3 was confirmed, and the results are shown in FIG. 2.

As shown in FIG. 2, Hydrogel Patch 2 was prepared in a liquid formulation in the same manner as in Example 1-1, and then immersed in a vial, and thrombin was immersed in a syringe in the same manner as in Example 1-3. . Hydrogel patch 2 was also lyophilized (at Scientific, F78) for 24 hours at −80 ° C., 0.33 Torr atmosphere to form a solid formulation, which was then prepared in powder form. In addition, formulations in powder form were prepared. The powder form formulation was gelled by adding thrombin in the same manner as in Example 1-3 to confirm that the powder formulation can be prepared in the form of gel again.

As a result, it can be seen that the hydrogel patch according to one embodiment may be provided in various formulations such as a reversible phase transition, such as a solid, semisolid, liquid or powder formulation.

Additionally, cryopreservation was confirmed.

Specifically, hydrogel patch 2 was added to 10% (v / v) DMSO by adding dimethyl sulfoxide (DMSO, Sigma, D2650) to the culture solution prepared in the same manner as in Example 1-2. Thereafter, freeze-preservation and freeze-drying were performed by sequentially lowering the temperature to 4 ° C (30 minutes), -20 ° C (1 hour), and -80 ° C. Thereafter, cold FE-Scanning Electron Microscopy (Cold FE-SEM; Hitach High-Technologies, S-4800) was used to perform morphological analysis of the hydrogel patch before and after cryopreservation. 3 is shown.

As shown in FIG. 3, there was no morphological difference between the thawed hydrogel patches before cryopreservation and 1 month after cryopreservation. It was also found that the surface of the hydrogel patch was porous.

<실험예 1> 시험관내 신경줄기세포 분화평가Experimental Example 1 In Vitro Neural Stem Cell Differentiation Evaluation

Brains were harvested at the expense of 5 day old C57BL / 6 mice (C57BL / 6N Japan SLC, Inc.), and mouse neural stem cells were cultured in a 100 mm dish (SARSTEDT, 831802) by primary cell culture using a known method.

By applying the material according to one embodiment and the comparative example material to the mouse neural stem cells, the effect on the differentiation of mouse neural stem cells was confirmed.

Specifically, hydrogel (fibrin) prepared in the same manner as in Example 1-2 with respect to extracting and culturing mouse neural stem cells by the known method (Kim JB et al. Nat Protoc. 2009), and for the 1x10 ⁴ neural stem cells. 5 mg / mL; 10 μg / ml laminin; and 0.5 mg / ml hyaluronic acid) were applied and thrombin added to make a hydrogel patch. Thereafter, the cells were cultured in a culture medium containing or without the neuronal growth factor and vascular endothelial growth factor of Example 1-2. In addition, to select the optimum concentration, as a comparative material, fibrin alone 1 mg / ml, 5 mg / mL or 10 mg / ml; Laminin alone 5 μg / ml, 10 μg / ml or 50 μg / ml; And 0.1 mg / ml, 0.5 mg / ml or 1 mg / ml of hyaluronic acid alone; Or 10 μg / ml laminin and 0.5 mg / ml hyaluronic acid.

Next, after 14 days of culture, immunofluorescence staining was performed. Specifically, for immunocytochemistry, cells were fixed with 4% (w / v) Paraformaldehyde in Phosphate Buffer Solution (Wako, 163-20145) at room temperature for 10 minutes. The fixed cells were diluted 3 × with 10X Phosphate Buffer Saline (PBS, P2007) and washed three times at room temperature for 5 minutes, and at room temperature using 0.1% (v / v) Triton X-100 (Sigma, T9284) dissolved in 1X PBS. Permeabilized for 10 minutes. After washing three times at room temperature with 1 × PBS for 5 minutes, blocking was performed for 1 hour at room temperature with 4% (v / v) fetal bovine serum dissolved in 1 × PBS to inhibit nonspecific binding. Cells were then incubated with primary antibody anti-beta III tubulin (Tuj1) (1: 400; Abcam) or anti-glial fibrillary acidic protein (GFAP) (1: 400; Sigma) for 1 hour at room temperature and three times in 1X PBS. Washed 0.05% (v / v) Tween-20 (Sigma, P7949) (PBST) was washed three times for 10 minutes at room temperature. Subsequently, light was blocked with a secondary fluorescent antibody (Alexa Fluorophore-conjugated secondary antibodies 488 (1: 1000) or Alexa Fluor® 594 (1: 1000) and incubated for 30 minutes at room temperature. Additional blocking was performed at room temperature for 30 minutes before incubating with other primary antibodies of 3 washes for 10 minutes at room temperature using PBST and then blocking light for 15 seconds in DAPI (1: 1000; Invitrogen) for cell nuclear staining. After incubation, the cells were washed three times for 10 minutes at room temperature using PBST Cells were stored in PBS for visualization using a fluorescence microscope.

Thereafter, neuronal cells (Tuj1) and astrocytes (GFAP) were observed using an inverted fluorescence microscope (Leica, DMI 3000B) attached with a digital monochrome camera (Leica, DFC345 FX). Images observed with inverted fluorescence microscopy were counted by counting the number of neurons or astrocytes using ImageJ (National Institutes of Health (http://rsb.info.nih.gov/ij) software).

All statistical analyzes were performed using the unpaired two-tailed Student's t-test with significance of * P <0.05, ** P <0.005 or *** P <0.0005.

As shown in FIG. 4, when the hydrogel was applied to the neural stem cells according to one embodiment, it was found that the cell junctions of the neural stem cells were improved as compared to the uncoated condition (bare).

5 and 6, when the composition (fibrin, laminin, hyaluronic acid) according to one embodiment is applied as compared to the case of applying the fibrin alone, laminin alone, hyaluronic acid alone, and a combination of laminin and hyaluronic acid as shown in FIG. It was found that differentiation of neural stem cells into neurons occurred synergistically. Specifically, it was found that 5% of neurons appeared in the control group treated with nothing, and less than 12% of the neurons appeared in each substance alone. In contrast, about 15% of neurons appeared in the combination of hyaluronic acid and laminin, and more than 20% of the neurons appeared when the hyaluronic acid and laminin were combined with fibrin, which had no effect alone. Could know. This means that the combination of hyaluronic acid and laminin has a remarkable effect of differentiation of neurons, and in particular, the combination of three compositions has a synergistic effect, significantly increasing differentiation into neurons.

In addition, as shown in FIG. 7, when the growth factor is added, fibrin alone, laminin alone, hyaluronic acid alone, and laminin and hyaluronic acid combinations are applied in accordance with the results of FIG. 6. When the composition (fibrin, laminin, hyaluronic acid) was applied, it was found that differentiation of neural stem cells into neurons occurred synergistically. Specifically, the growth factor itself increases the differentiation into neurons, but produces a synergistic effect by the combination of the three compositions and growth factors, resulting in about 30% of the neurons. It is meant that hydrogel patches or compositions accordingly exhibit a synergistic effect that is typically unpredictable.

In addition, as shown in Figure 8, by adding each growth factor to the composition according to one embodiment as a result, it was confirmed that the rate of differentiation of mouse neural stem cells into neurons about 2.5 to 5 times.

As a result of the above, it can be seen that the composition according to one embodiment has not only a synergistic effect of increasing the degree of differentiation on its own, but also shows a more synergistic effect in combination with growth factors, and increases the effect of growth factors on cells. there was.

<실험예 2> 생분해성 평가Experimental Example 2 Biodegradability Evaluation

In order to evaluate the biodegradability of the hydrogel patch prepared in Example 1, 2,2,2-tribromoethanol (Sigma, T48402) was added to Tert-amyl alcohol (C57BL / 6N Japan SLC, Inc.). 25 mg / ml Avertin anesthetics prepared by dissolving in Sigma, 152463) were administered at 125-250 mg / kg body weight per mouse. Thereafter, hydrogel patch 5 was implanted subcutaneously. Two weeks after the gel patch was implanted, subcutaneous dissection was performed to confirm the biodegradation of the implanted gel patch.

As a result, as shown in FIG. 9, the hydrogel patch according to one embodiment was confirmed to be biodegradable in vivo.

<실험예 3> 하이드로젤 패치 척수손상 치료효과 확인Experimental Example 3 Hydrogel Patch Spinal Cord Injury Treatment Effect Confirmation

4-1. 척수손상동물 모델을 이용한4-1. Using Spinal Cord Injured Animal Model 하이드로젤 패치 척수손상 치료효과 확인Hydrogel patch spinal cord injury treatment effect confirmed

To confirm the spinal cord injury regeneration effect of the hydrogel patch according to one embodiment, behavioral evaluation was performed for 8 weeks after the gel patch was implanted in the spinal cord injury animal model.

The official small-scale spinal cord injury animal model evaluation method evaluates the behavioral analysis of the study of chronic spinal cord injury studies. Observation and evaluation to evaluate the recovery of behavioral ability. Reflecting this, each animal is scored from 0 to 21 to quantify its resilience, and 0 to 7 represents early stage recovery of minimal behavioral ability with almost no recovery of hind limb movement. Up to 13, the intermediate recovery stage (intermediate recovery stage) has recovered the movement of the hind limbs, but there is a gap between harmonic movements with poor control of the front and hind legs. Finally, 14 to 21 are late recovery stages, which represent the harmonization of the front and hind legs.

First, adult male Sprague-Dawley rats (OrientBio) were anesthetized by injecting 10 mg / rat Zoletil 50 (Virbac), and the spinal cord constriction of the T9 portion was removed. Then, spinal cord was pressed for 10 minutes using a vascular clip to produce a spinal cord injury animal model. After spinal cord injury, urine management was performed by pressing the bladder twice a day for about two weeks until spontaneous urination was possible. One week after the spinal cord injury animal model was prepared, a hydrogel patch according to one embodiment was directly implanted into the spinal cord injury site as shown in FIG. 10. Behavioral evaluation was performed using the spinal cord injury animal model evaluation method (BBB test; open field test) for 8 weeks, and the test results were obtained using the mean values of observations of the two examiners, and the results are shown in FIGS. 15 to 18.

11 is a view of the hind limb movement of the spinal cord injury animal model lost to spinal cord injury after one week of spinal cord injury animal model. 12 is a view of the hind limb movement of the spinal cord injury week 8 of the spinal cord injury animal model sprayed only PBS on the implantation site without implantation of the hydrogel patch. FIG. 13 is a diagram illustrating hind limb movement at 8 weeks of spinal cord injury in a spinal cord injury animal model implanted with Hydrogel patch 2 without GF. FIG. 14 is a diagram illustrating hind limb movement at week 8 of spinal cord injury in a spinal cord injury animal model implanted with Hydrogel patch 5 (GF).

15 to 18 are graphs showing the BBB score values according to the implantation of the hydrogel patch according to one embodiment.

As shown in FIGS. 11 to 14, the spinal cord injury animal model in which only PBS was applied to the implanted site without the implantation of the hydrogel patch was found to have no change in the motility of the damaged hind limb after 8 weeks of spinal cord injury. In contrast, the spinal cord injury model implanted with hydrogel patch 2 recovered the motility of the left hind limb after 8 weeks of spinal cord injury, and the spinal cord implanted with hydrogel patch 5 added with neuronal growth factor and vascular endothelial growth factor. The injured animal model confirmed that the motor capacity of both hind limbs was significantly restored after 8 weeks of spinal cord injury.

In addition, as a result of evaluating spinal cord injury recovery ability using a spinal cord injury animal model evaluation method (BBB test; open field test), as shown in Figs. 15 to 18, only PBS is applied to the implantation site without the hydrogel patch implantation. Spinal cord injury model showed a BBB score of 4-6. This represents the early stage of the spinal cord injury regeneration process. In contrast, the spinal cord injury animal model implanted with hydrogel patch 2 showed a BBB score of about 8 to 10, indicating an intermediate recovery stage of the spinal cord injury regeneration process. In addition, the spinal cord injury animal model implanted with hydrogel patch 5 showed a BBB score of about 10 to 12, indicating that it is in the late recovery phase of the spinal cord injury regeneration process.

15-18, spinal cord injury animal models implanted with

hydrogel patches

1 and 4 show BBB scores between about 6 and 8 indicating early stages of the spinal cord injury regeneration process. Could. The above results mean that the hydrogel patch containing fibrin, laminin and hyaluronic acid showed a remarkable effect compared to other combinations.

In addition, as a result of comparing the hydrogel patch 2, and the hydrogel patch 5 and the

hydrogel patches

3 and 6 to which collagen was added, it was found that the BBB score was not significantly different.

In addition, when comparing the hydrogel patch 3 and the addition of only PBS to the hydrogel patch 3, it can be seen that there is no significant difference in the BBB score, through which the cell culture medium does not play a role in the hydrogel patch efficacy Could.

Accordingly, the results showed that the hydrogel patch according to the embodiment has a therapeutic effect that remarkably restores the motor ability lost due to spinal cord injury.

4-2. 조직학적 분석를 이용한 하이드로젤 패치 척수손상 치료효과 확인4-2. Therapeutic Effect of Hydrogel Patch Spinal Cord Injury Using Histological Analysis

To confirm the spinal cord injury regeneration effect of the hydrogel patch according to one embodiment, histological analysis was performed.

Specifically, rats were sacrificed 8 weeks after hydrogel patch transplantation for histological analysis of the spinal cord injury nerve regeneration effect of hydrogel patch, and then perfused with 4% (w / v) paraformaldehyde (Merck) to spinal cord. Was taken. Thereafter, the spinal cord was cut to a thickness of 10 μm, and the cut spinal cord sample was subjected to anti-neurofilament (NF) (1: 3000, Abcam), anti-GFAP (1: 1000, Abcam), and 2 ', 3'-Cyclic-nucleotide 3'-phosphodiesterase (CNPase) (1: 800, Abcam), anti-Homeobox HB9 (Hb9) (1: 100, DSHB), anti-ionized calcium-binding adapter molecule 1 ( Iba-1) (1: 500, abcam). Thereafter, by confocal microscopy (LSM 700, Zeiss) to evaluate the neuronal regeneration of the hydrogel implantation site, the results are shown in Figure 19.

As a result, as shown in FIG. 19, when

hydrogel patch

2 or 5 was implanted as compared to the case where PBS was applied to the implanted site, the distribution of GFAP astrocytes in the spinal cord injury lesion area was low, whereas the distribution of NF neurons Was found to be high. In addition, when the

hydrogel patch

2 or 5 is implanted, it was found that CNPase oligodendrocytes are co-localized at the same position as NF neurons. These results indicate that the regeneration of damaged neurons and myelin formation of regenerated neurons was significantly increased by the application of hydrogel patches.

In addition, the distribution of NF neurons and the distribution of Iba-1 microglia, which exhibit an inflammatory response, were low when PBS was applied to the implanted sites, whereas Hb9 and NF of the spinal cord were implanted when

hydrogel patches

2 or 5 were implanted. It was confirmed that the distribution of motor neurons expressing with and the distribution of Iba-1 microglia was significantly reduced. These results indicate that the inflammatory response caused by spinal cord injury during hydrogel patch transplantation inhibits nerve damage, and helps regeneration of motor nerve cells and oligodendrocytes, thereby regenerating motor nerve cells with myelin formation. .

As a result of this, the hydrogel patch has a marked increase in the therapeutic effect of restoring lost motor ability by inhibiting the induction of damaged spinal nerve regeneration and activation of astrocytes, and spinal cord 2 caused by spinal cord injury treatment using a syringe. It was found that there is an effect that can significantly inhibit the car damage by using a hydrogel patch.

Claims

Fibrin or, fibrin and fibrinogen;

Laminin; And

Hydrogel patch comprising hyaluronic acid or a salt thereof.
The hydrogel patch of claim 1, further comprising a cell growth factor.
The method of claim 2, wherein the cell growth factor, neuronal growth factor, vascular endothelial growth factor, fibroblast growth factor, bone morphogenetic protein, epidermal growth factor, epidermal growth factor, hepatocyte growth factor, trait Hydrogel patch, which is a conversion growth factor or a combination thereof.
According to claim 3, wherein the neuronal growth factor BDNF (Brain-derived neurotropic factor), GDNF (Glial cell derived neurotropic factor), CNTF (Ciliary neurotropic factor), bFGF (basic fibroblast growth factor), cAMP (cyclic adenocyne monophosphate ), NT (Neurotropin), NT3 (Neurotropin-3), NT4 (Neurotropin-4), T3 (Triiodo-L-Thyronine), SHH (Sonic hedgehog), and PDGF (Platelet-derived growth factor) Hydrogel patch containing at least one.
The hydrogel patch of claim 3, wherein the vascular endothelial growth factor is VEGF (vascular endothelial growth factor).
The hydrogel patch of claim 1, wherein the hydrogel patch does not comprise cells.
The hydrogel patch of claim 1, having a porosity on the surface.
The hydrogel patch of claim 1, wherein reversible phase transition occurs in a solid, semisolid, or liquid state with temperature.
The hydrogel patch of claim 1, wherein the hydrogel patch is applied to a tissue injury site and modified in shape to match the shape of the injury site.
The hydrogel patch of claim 1, for regeneration or repair of damaged tissue.
The hydrogel patch of claim 10, wherein the damaged tissue is a spinal cord.
The hydrogel patch of claim 10, wherein the damaged tissue is chronic spinal cord injury.
The method of claim 1, comprising fibrin or fibrinogen at a concentration of 0.5 to 20 mg / ml, laminin at a concentration of 1 to 100 μg / ml, or hyaluronic acid at a concentration of 10 μg / ml to 5 mg / ml, Hydrogel patch.
The hydrogel patch of claim 1, wherein the hydrogel patch is usable by non-invasive spinal cord at the site of injury of a spinal cord injury patient.
The hydrogel patch of claim 1, further comprising thrombin.
Fibrin or, fibrin and fibrinogen;

Laminin; And

A pharmaceutical composition for treating spinal cord injury, comprising hyaluronic acid or a pharmaceutically acceptable salt thereof.
The pharmaceutical composition of claim 16, further comprising a cell growth factor.
The method according to claim 17, wherein the cell growth factor, neuronal growth factor, vascular endothelial growth factor, fibroblast growth factor, bone morphogenetic protein, epidermal growth factor, hepatocyte growth factor, It is a transforming growth factor or a combination thereof.
The method of claim 17, wherein the neuronal growth factor is Brain-derived neurotropic factor (BDNF), Glial cell derived neurotropic factor (GDNF), Ciliary neurotropic factor (CNTF), basic fibroblast growth factor (bFGF), cyclic adenocyne monophosphate ), NT (Neurotropin), NT3 (Neurotropin-3), NT4 (Neurotropin-4), T3 (Triiodo-L-Thyronine), SHH (Sonic hedgehog), and PDGF (Platelet-derived growth factor) It will contain at least one.
The pharmaceutical composition according to claim 16, wherein the composition has porosity on the surface when the composition is solid or semisolid.
The pharmaceutical composition of claim 16, which is a powder formulation.
The pharmaceutical composition of claim 16, which is for the regeneration or repair of damaged tissue.
The method of claim 16, wherein the fibrin or fibrinogen comprises 0.5-20 mg / ml, laminin 1-100 μg / ml, or hyaluronic acid 10 μg / ml-5 mg / ml. Pharmaceutical compositions.
The pharmaceutical composition of claim 16, further comprising thrombin.
The pharmaceutical composition of claim 16, wherein the spinal cord injury is chronic spinal cord injury.
Fibrinogen;

Laminin; And

A process for preparing a hydrogel patch by adding thrombin to a composition in a sol state comprising hyaluronic acid or a pharmaceutically acceptable salt thereof.
27. The method of claim 26, wherein the composition of the sol state neuronal growth factor, vascular endothelial growth factor, fibroblast growth factor, bone morphogenetic protein, epidermal growth factor, epidermal growth factor, hepatocyte growth factor Further comprising the step of combining the transforming growth factors or combinations thereof.
The method of claim 26, comprising cryopreserving or cryopreserving the hydrogel patch at 4 to -210 ° C. in solution.