Gel, manufacturing method therefor, and use thereof

Abstract

A gel, comprising powder particles, β-tricalcium phosphate, and a carrier, wherein the powder particles comprise glutamine transaminase, gelatin, and sodium polyacrylate. Glutamine transaminase is covalently cross-linked with gelatin, and glutamine transaminase is covalently cross-linked with sodium polyacrylate. A method for manufacturing the gel, comprising: mixing glutamine transaminase, gelatin, and sodium polyacrylate to obtain a reaction product; freeze-drying and grinding the reaction product to obtain powder particles; and mixing the powder particles, β-tricalcium phosphate, and a carrier to obtain the gel. Use of the gel for preparing a pharmaceutical composition for promoting tissue repair or regeneration.

Description

Gel, method for producing the same and use thereof Technical Field

[0001] The present disclosure relates to a gel, a method for manufacturing the same, and uses thereof, and more particularly to a gel for promoting tissue repair or regeneration. Background Art

[0002] Vertebrate tissues, including bone, often suffer from defects due to trauma, aging, degeneration, osteoporosis, and bone tumor resection. In conventional bone repair techniques, bone cement, due to its high mechanical strength, is used to fix metal implants and fill tissue defects.

[0003] However, the high temperatures generated during the curing process of bone cement can damage surrounding tissues. During the sharing of bone cement and metal implants, bone cement not only fails to reshape the bone but can even destroy it when subjected to external forces. This fails to effectively address issues such as prosthetic loosening, periprosthetic osteolysis, poor osteoconductivity and osteoinduction, and incomplete bone healing after the use of bone cement. Furthermore, the liquid component of PMMA bone cement has been shown to be cytotoxic.

[0004] In addition, if hydrogel materials are injected into the affected area, the colloid will often swell due to excessive water absorption, which will cause swelling or pain in the affected area, excessive overflow of the colloid to areas outside the affected area, or implant shedding.

[0005] Therefore, there is still an urgent need in the art for a gel that can solve the above problems, and a preparation method and use thereof. Summary of the Invention

[0006] In view of the above problems, the present disclosure provides a gel comprising granules and a carrier, wherein the granules comprise a reaction product of transglutaminase, gelatin and sodium polyacrylate.

[0007] The present disclosure further provides a method for producing a gel, comprising: providing a solution containing transglutaminase and a mixture of gelatin and sodium polyacrylate; mixing the solution and the mixture to obtain a reaction product; freeze-drying and grinding the reaction product to obtain a powder; and mixing the powder, β-tricalcium phosphate, and a carrier to obtain a gel.

[0008] The present disclosure also provides a use of the gel for preparing a pharmaceutical composition for promoting tissue repair or regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figures 1 to 4 are schematic diagrams of the preparation method of the gel according to at least one embodiment of the present disclosure. Figure 5 is a scanning electron microscope (SEM) photograph of the gel according to at least one embodiment of the present disclosure. Figure 6 is a graph showing the results of a cytotoxicity test of the gel according to at least one embodiment of the present disclosure. Figure 7 is a graph showing the temperature-time results of the bone cement forming process of the gel according to at least one embodiment of the present disclosure and a comparative example. The forming temperature was measured using a thermocouple thermometer. Figure 8 is a computed tomography (CT) photograph of the gel according to at least one embodiment of the present disclosure and the bone cement of the comparative example used as a filler for a rabbit ankle defect. Figure 9A is a CT photograph of the gel according to at least one embodiment of the present disclosure and the bone cement and bone powder of the comparative example used as a filler for a pig tibial defect. Figure 9B is a graph showing the results of a pull-out test of implants according to at least one embodiment of the present disclosure and a control group. Figure 10A is a confocal microscope photograph of bone cell mineralization according to at least one embodiment of the present disclosure. Figure 10B is a histogram of the amount of bone cell mineralization according to at least one embodiment of the present disclosure. *P-value <0.0001 (compared to day 0). Figure 11 is a drug release curve diagram of the gel according to at least one embodiment of the present disclosure. Figure 12A is a confocal microscope photograph of bone cell mineralization according to at least one embodiment of the present disclosure. Figure 12B is a histogram of the amount of bone cell mineralization according to at least one embodiment of the present disclosure. *P-value <0.05 (compared to the negative control group). Figure 13 is a CT photograph of bone defect healing in mice 29 days after surgery according to at least one embodiment of the present disclosure. Figure 14 is a schematic structural diagram of the gel according to at least one embodiment of the present disclosure and at least one comparative example. Figure 15 is an SEM photograph of the gel according to at least one embodiment of the present disclosure and at least one comparative example. Figure 16 is an SEM photograph of gelatin according to at least one preparation example of the present disclosure. Figure 17 is an SEM photograph of powder and particles according to at least one preparation example of the present disclosure. Implementation

[0010] The following examples are provided to illustrate the present disclosure in detail. A person skilled in the art, after reading the disclosure of this specification, can readily understand the advantages and effects of the present disclosure, and can also implement or apply it in other different embodiments. Therefore, the following embodiments for implementing the present disclosure can be modified and / or changed without violating the scope of its various aspects and applications, and any element or method within the scope of the present disclosure can be combined with any other element or method disclosed in any embodiment of the present disclosure.

[0011] As used herein, the articles “a”, “an”, “the” and “said” refer to one or more than one (i.e., to at least one) of the grammatical object of the article, unless expressly stated otherwise, and “or”, “ / ” and “and / or” are used interchangeably unless expressly stated otherwise.

[0012] As used herein, the phrase "at least one" refers to one or more elements and should be understood to mean at least one element selected from any one or more of the listed elements, but does not necessarily include at least one of every listed element in the listed elements and does not exclude any combination of elements in the listed elements. This definition also allows for the optional presence of elements other than the elements identified in the list of elements to which the phrase "at least one" refers, whether related or unrelated to the identified elements. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B", or, equivalently, "at least one of A and / or B") may refer in one embodiment to at least one A, optionally including more than one A, and no B (and optionally including elements other than B); in another embodiment, to at least one B, optionally including more than one B, and no A (and optionally including elements other than A); and in yet another embodiment, to at least one A, optionally including more than one A, and at least one B, optionally including more than one B (and optionally including other elements).

[0013] As used herein, the terms "include," "comprising," "containing," "having," and any other variations thereof are intended to cover a non-exclusive inclusion. For example, when describing something as "including" a limitation, unless otherwise stated, it may additionally include other ingredients, components, parts, structures, regions, parts, devices, systems, steps, or connections, and other limitations should not be excluded.

[0014] The ordinal numbers referred to in this disclosure, such as "first" and "second", do not represent the order of a component or feature relative to another component or feature, or the order in the manufacturing method. Such ordinal numbers are only used to modify the components or features so as to clearly distinguish certain components or features with the same name.

[0015] The numerical ranges used herein are inclusive and combinable, and any numerical value falling within the numerical range herein can be regarded as a maximum or minimum value to derive a subrange therefrom. For example, the numerical range "10 to 30 wt %" includes any subrange between a minimum of 10 wt % and a maximum of 30 wt %, such as a subrange from 10 to 15 wt %, 22 to 30 wt %, 17 to 25 wt %, etc. In addition, a plurality of numerical values ​​used herein can be selected as maximum and minimum values ​​as needed to derive a numerical range. For example, the numerical ranges of 2 to 5 wt %, 2 to 10 wt %, and 5 to 10 wt % can be derived from the numerical values ​​of 2 wt %, 5 wt %, and 10 wt %.

[0016] The term "about" as used herein refers to a range that is within the typical tolerance range in the art. For example, "about" can be understood to mean about 2 standard deviations from the mean. When "about" appears before a series of numbers or a range, it should be understood that "about" can modify each number in the series or range. For example, a value is intended to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or ±0.1% from the value. As used herein, numerical ranges are inclusive and combinable, and any value falling within a numerical range herein can be considered a maximum or minimum value from which subranges can be derived. For example, it should be understood that the numerical range "20 to 30%" includes any subrange between a minimum of 20% and a maximum of 30%, such as subranges from 20 to 25%, from 25 to 30%, and from 22.5 to 27.5%. Such variations in numerical values ​​may occur due to, for example, experimental error, typical errors in measurement or handling in manufacturing compounds, compositions, concentrates, or formulations, differences in the origin, manufacture, or purity of starting materials or ingredients used in the present disclosure, or similar considerations. Alternatively, the term "about" means within an acceptable standard deviation of the mean as considered by one of ordinary skill in the art. Unless expressly stated otherwise, all numerical ranges, amounts, values, and percentages disclosed herein, such as amounts of material, duration of time periods, temperatures, operating conditions, ratios of amounts, and the like, should be understood to be modified in all instances by the term "about."

[0017] As used herein, the term "treat" refers to obtaining a desired pharmacological or physiological effect, such as partially or completely preventing, ameliorating, alleviating, or controlling a symptom, condition, or disorder associated with a disease. As used herein, the term "treat" may refer to applying or administering at least one therapeutic agent or method to a subject exhibiting a symptom, condition, or disorder associated with a disease, with the goal of partially or completely ameliorating, alleviating, relieving, relieving, delaying the onset of, reducing the severity of, inhibiting the progression of, or reducing the incidence of at least one symptom, condition, or disorder associated with the disease. In some embodiments, treatment may be administered to a subject exhibiting only early signs of such a symptom, condition, or disorder associated with the disease, with the goal of reducing the risk of developing the same disease, although the present disclosure is not limited thereto.

[0018] As used herein, the terms "subject" and "patient" are interchangeable and refer to animals, such as mammals. The term "subject" is intended to refer to both males and females, unless a gender is specifically designated. In at least one embodiment of the present disclosure, the subject is a rodent, mouse, monkey, dog, cat, cow, horse, emu, sheep, deer, wolf, fox, pig, rabbit, chicken, ostrich, or human, but the present disclosure is not limited thereto. In some embodiments, the subject is human.

[0019] As used herein, the term "administer" refers to the process of introducing an active ingredient into a subject by a method or route such that at least a portion of the active ingredient is localized to a desired site to produce a desired effect. For example, the active ingredients of the present disclosure may be administered to a subject by injection or topical administration, but the present disclosure is not limited thereto. Administration of the compositions of the present disclosure may be performed systemically or locally within a subject. For example, the site of topical administration may be any site in the body where desired or beneficial tissue development is desired, such as a joint, a surgical site, a site of non-healing tissue, a wound, or an ulcer.

[0020] As used herein, the term "carrier" can be water, deionized water, saline, or a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier can be, for example, a liquid or solid filler, diluent, solvent, or encapsulating material. In some embodiments, each component is "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the cosmetic or pharmaceutical formulation and suitable for use in contact with tissues or organs of a subject (e.g., a human or animal) without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications commensurate with a reasonable benefit / risk ratio. See Remington: The Science and Practice of Pharmacy, 22nd ed.; Allen, ed.: Philadelphia, PA, 2012; Handbook of Pharmaceutical Excipients, 7th ed.; Rowe et al., eds.; Pharmaceutical Press and American Pharmaceutical Association: 2012; Handbook of Pharmaceutical Additives, 3rd ed.; Ash and Ash, eds.; Gower Publishing Company: 2007; Pharmaceutical Preformulations and Formulations, 2nd ed.; Gibson, ed.; CRC Press LLC: Boca Raton, FL, 2009.

[0021] As used herein, the term "drug" refers to a substance that produces a biological effect on an individual or patient, and is used to alter the physiological and / or metabolic functions of the individual or patient, thereby achieving the medical purpose of preventing, treating, diagnosing and / or ameliorating a disease.

[0022] As described herein, "cell preparation" refers to a cell product used to reconstruct, repair, replace, support, improve or treat the structure or function of human tissues. In at least one embodiment of the present disclosure, the cell preparation can achieve the effect of treating or preventing diseases, and the applicable clinical treatment fields include but are not limited to: internal medicine, cardiology and vascular medicine, dermatology, otolaryngology, neurology, orthopedics, ophthalmology or cancer-related. In at least one embodiment of the present disclosure, the cell preparation includes but is not limited to: genetically engineered autologous cells (such as but not limited to chimeric antigen receptor T cells (Car-T cells)) or allogeneic cells, stem cells, immune cells, fibroblasts, chondrocytes, osteocytes, osteoblasts, osteoclasts, or cell derivatives. In some embodiments of the present disclosure, the stem cells may be multipotent stem cells, pluripotent stem cells, adult stem cells, tissue-specific stem cells, or mesenchymal stem cells, such as, but not limited to, peripheral blood stem cells, adipose stem cells, bone marrow mesenchymal stem cells, cartilage stem cells, or limbal stem cells. In some embodiments of the present disclosure, derivatives of the above cells may be exosomes, secretomes, conditioned medium, or extracellular vehicles, but the present disclosure is not limited thereto. In some embodiments of the present disclosure, the above cells may be autologous cells or allogeneic cells, such as, but not limited to, autologous peripheral blood stem cells, autologous adipose stem cells, autologous bone marrow mesenchymal stem cells, autologous cartilage stem cells, autologous chondrocytes, autologous immune cells, autologous fibroblasts, or allogeneic limbal stem cells.

[0023] As used herein, the term "remaining amount of carrier" refers to an amount of carrier included such that the sum of the components in the gel is 100 weight percent (wt%) based on the total weight of the gel.

[0024] As used herein, the term "osteointegration" refers to the direct attachment or apparent connection of bone tissue to an allogeneic inert material without the presence of connective tissue intrusion between the bone tissue and the allogeneic inert material.

[0025] As used herein, the term "osteoconduction" refers to guiding osteoprogenitor cells to grow from a bone bed into a three-dimensional porous environment to fill in the gaps in bone tissue.

[0026] As used herein, the term "osteoinduction" refers to the stimulation of primitive, undifferentiated multipotent cells to develop into bone-forming cell lineages.

[0027] As described in this article, the term "stress shielding" refers to the phenomenon of uneven stress transfer caused by a significant difference in Young's modulus between the implant and native bone. Stress shielding leads to poor osseointegration and, in turn, additional bone damage.

[0028] In at least one embodiment of the present disclosure, the powder and granules may comprise approximately 10 to 25 wt %, for example, but not limited to, 11 to 24 wt %, 12 to 23 wt %, or 13 to 22 wt %, based on the total weight of the gel. In some embodiments, the powder and granules may comprise approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 wt %, but the present disclosure is not limited thereto.

[0029] In at least one embodiment of the present disclosure, the content of β-tricalcium phosphate may be about 2 to 10 wt %, such as, but not limited to, 2.5 to 9.5 wt %, 3 to 9 wt %, or 3.5 to 8.5 wt %, based on the total weight of the gel. In some embodiments, the content of β-tricalcium phosphate may be about 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 wt %, but the present disclosure is not limited thereto.

[0030] In at least one embodiment of the present disclosure, the gel may include a residual amount of carrier, drug and / or cell preparation based on the total weight of the gel. In some embodiments, the drug and / or cell preparation may be dissolved in the carrier to form a solution, such as but not limited to an aqueous solution.

[0031] In at least one embodiment of the present disclosure, the weight ratio of the powder and β-tricalcium phosphate to the carrier, or the weight ratio of the powder and β-tricalcium phosphate to the carrier and the drug, may be 1:2 to 3, for example, but not limited to, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, or 1:3, but the present disclosure is not limited thereto. In some embodiments, the above-mentioned weight ratios of the drug to other ingredients are also applicable to cell preparations.

[0032] In at least one embodiment of the present disclosure, the carrier may be water, deionized water, physiological saline, or a pharmaceutically acceptable carrier.

[0033] In at least one embodiment of the present disclosure, β-tricalcium phosphate may be in the form of crystals or powder.

[0034] In at least one embodiment of the present disclosure, sodium polyacrylate may be an aqueous solution or a powder.

[0035] In at least one embodiment of the present disclosure, the transglutaminase may be an aqueous solution of transglutaminase.

[0036] In at least one embodiment of the present disclosure, the gelatin can be in the form of a colloid or a powder. In some embodiments, the gelatin powder can be obtained by freeze-drying and grinding gelatin.

[0037] In at least one embodiment of the present disclosure, the particle size of the gelatin powder may be approximately 850 μm or less, for example, but not limited to, 1 to 850 μm, 10 to 840 μm, or 20 to 830 μm. In some embodiments, the particle size of the gelatin powder may be approximately 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, or 850 μm, but the present disclosure is not limited thereto.

[0038] In at least one embodiment of the present disclosure, the weight ratio of transglutaminase, gelatin, and sodium polyacrylate is 4-6:3-5:1.

[0039] In at least one embodiment of the present disclosure, the weight ratio of transglutaminase to sodium polyacrylate may be about 4:1 to 6:1, for example, but not limited to, 4:1, 5:1, or 6:1. In some embodiments, the weight ratio of transglutaminase to sodium polyacrylate may be about 4:1, 4.1:1, 4.2:1, 4.3:1, 4.4:1, 4.5:1, 4.6:1, 4.7:1, 4.8:1, 4.9:1, 5:1, 5.1:1, 5.2:1, 5.3:1, 5.4:1, 5.5:1, 5.6:1, 5.7:1, 5.8:1, 5.9:1, or 6:1, but the present disclosure is not limited thereto.

[0040] In at least one embodiment of the present disclosure, the weight ratio of gelatin to sodium polyacrylate may be about 3:1 to 5:1, for example, but not limited to, 3:1, 4:1, or 5:1. In some embodiments, the weight ratio of gelatin to sodium polyacrylate may be about 3:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4:1, 4.1:1, 4.2:1, 4.3:1, 4.4:1, 4.5:1, 4.6:1, 4.7:1, 4.8:1, 4.9:1, or 5:1, but the present disclosure is not limited thereto.

[0041] In at least one embodiment of the present disclosure, the powder, β-tricalcium phosphate, and the carrier interact to form a construct, and transglutaminase can promote a cross-linking reaction between gelatin and a portion of the OH groups of sodium polyacrylate to form a covalent bond. The calcium ions of β-tricalcium phosphate react with the carboxyl groups of the sodium polyacrylate, and another portion of the OH groups of the sodium polyacrylate forms hydrogen bonds with the carrier.

[0042] In at least one embodiment of the present disclosure, sodium polyacrylate is pre-crosslinked and calcium ions of β-tricalcium phosphate (i.e., Ca 2+ ) is bonded to the carboxyl groups of cross-linked sodium polyacrylate to enhance the structural strength and stability of the gel, thereby preventing the gel from disintegrating in body fluids, thus overcoming the problem of conventional bone cements (such as calcium-phosphate bone cements) disintegrating in body fluids.

[0043] In at least one embodiment of the present disclosure, the reaction product may have a sponge-like structure, which may include a plurality of interconnected pores.

[0044] In at least one embodiment of the present disclosure, the sponge-like structure may further include a sheet-like structure.

[0045] In at least one embodiment of the present disclosure, the particle size of the powder is 850 μm or less, for example, but not limited to, 1 to 850 μm, 10 to 840 μm, or 20 to 830 μm. In some embodiments, the particle size of the powder may be approximately 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, or 850 μm, but the present disclosure is not limited thereto.

[0046] In at least one embodiment of the present disclosure, the gel may further contain drugs and / or cell preparations.

[0047] In at least one embodiment of the present disclosure, the drug may include at least one selected from the group consisting of anticancer agents, anti-inflammatory agents, protein drugs, antibiotics, platelet-rich plasma, and platelet-rich fibrin, or a combination thereof.

[0048] In at least one embodiment of the present disclosure, the protein drug may comprise at least one selected from the group consisting of epidermal growth factor, leukocyte growth factor, erythrocyte growth factor, neurotrophic factor and bone growth factor, or a combination thereof.

[0049] In at least one embodiment of the present disclosure, the bone growth factor comprises at least one member or combination thereof selected from the group consisting of bone morphogenetic protein, transforming growth factor-β (TGF-β), fibroblast growth factor (FGF), insulin-like growth factor (IGF), and vascular endothelial growth factor (VEGF). In some embodiments, the bone growth factor may be bone morphogenetic protein 2 (BMP2).

[0050] In at least one embodiment of the present disclosure, the gel may have a Young's modulus of less than 1000 MPa, such as, but not limited to, 0.5 to 1000 MPa, 0.6 to 500 MPa, or 0.7 to 100 MPa. In some embodiments, the Young's modulus may be about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 MPa, but the present disclosure is not limited thereto.

[0051] In at least one embodiment of the present disclosure, the gelatin may be in the form of gelatin powder.

[0052] In at least one embodiment of the present disclosure, when the gelatin is in the form of gelatin powder, the method for producing the gel further includes: providing a solution containing colloidal gelatin; and freeze-drying and grinding the solution containing the colloidal gelatin to obtain gelatin powder.

[0053] In at least one embodiment of the present disclosure, freeze drying can be used to form pores, and grinding can be used to form a powder with a specific particle size. In some embodiments, freeze drying and grinding can form a porous cross-linked structure.

[0054] In at least one embodiment of the present disclosure, the method for producing a gel comprising mixing a powder, β-tricalcium phosphate, and a carrier includes: storing the carrier in a first syringe and storing the powder and β-tricalcium phosphate in a second syringe; connecting the first syringe and the second syringe via a connector to thereby communicate with each other; and simultaneously pushing the piston assembly of the first syringe and the piston assembly of the second syringe back and forth to obtain a gel.

[0055] In at least one embodiment of the present disclosure, when the first syringe is not connected to the second syringe, the piston assembly of the first syringe or the piston assembly of the second syringe is used to push the gel out of the first syringe or the second syringe.

[0056] In at least one embodiment of the present disclosure, the syringe may be a syringe.

[0057] In at least one embodiment of the present disclosure, the syringe may include a long axis direction X.

[0058] In at least one embodiment of the present disclosure, the connector may be a two-way valve. In some embodiments, both ends of the two-way valve may include Luer connectors. In some embodiments, the Luer connectors may include threaded portions, thereby enabling the first and second syringes connected via the threaded portions to withstand greater pushing pressure when the first and second piston assemblies reciprocate.

[0059] In at least one embodiment of the present disclosure, the tissue may be tissue other than teeth. In some embodiments, the tissue may be bone tissue.

[0060] In at least one embodiment of the present disclosure, a pharmaceutical composition for treating bone defects in a subject in need thereof is provided, comprising a gel.

[0061] In at least one embodiment of the present disclosure, a method for treating bone defects is provided, comprising: administering an effective amount of a gel to a subject in need thereof.

[0062] In at least one embodiment of the present disclosure, the gel has a lower Young's modulus than native bone tissue, which can reduce stress shielding. The gel's porous structure and three-dimensional network structure allow bone cells to reside, thereby promoting bone integration and bone conduction, helping to improve the prognosis of osteoporosis patients. Therefore, the gel of the present disclosure can replace traditional bone cement and be applied to regular or irregular bone defects. It promotes stable and secure attachment of implants to bone tissue, preventing secondary fractures. Its osteoconductive properties allow cells to grow from native tissue areas and form new bone tissue, promoting bone remodeling and regeneration. Furthermore, the gel of the present disclosure can be used in combination with antibiotics to treat osteomyelitis. Example

[0063] Preparation Example 1-1. Gelatin Powder

[0064] Pour 400 mL of deionized water into a 1000 mL glass graduated cylinder. Then, pour the 400 mL of deionized water into a 1000 mL glass beaker. Use a heated stirrer to adjust the temperature of the 400 mL of deionized water, and use a thermometer to confirm that the deionized water has reached 50-60°C. Place a magnetic stir bar in the 400 mL of deionized water and adjust the stirring speed to 300-400 rpm.

[0065] Using an electronic scale, weigh 100 g of gelatin on a plastic weighing plate. Slowly add 100 g of gelatin in batches to a 1000 mL glass beaker containing 400 mL of deionized water to obtain a solution with a gelatin concentration of 20 wt%.

[0066] Stir the solution until the gelatin is completely dissolved. Using an electric pipette, draw 20 mL of the solution from a 1000 mL glass beaker and pour it into a silicone ice cube tray. Let it sit until the solution cools to room temperature. Set the refrigerator temperature to -20°C and freeze the solution in the freezer for 16 to 24 hours to obtain frozen gelatin.

[0067] The freeze dryer was turned on, the temperature was lowered to about -80°C and the pressure was lowered to about 1.3 Torr, and the frozen gelatin was freeze-dried for 72 hours. The pressure was then released and the freeze dryer was turned off to obtain freeze-dried gelatin.

[0068] Grind the freeze-dried gelatin in a grinder. Sieve the ground gelatin through a 20-mesh sieve to obtain gelatin granules with a particle size of approximately 850 μm or less. Dry and store the gelatin granules in a 50 mL centrifuge tube.

[0069] Preparation Example 1-2. Gelatin Granules

[0070] As the gelatin particles of Preparation Example 1-2, gelatin that had not been freeze-dried and ground was used.

[0071] Preparation Example 2. Preparation of powder and granules

[0072] In a 50 mL centrifuge tube, add normal saline, 5 g of transglutaminase, and normal saline in order to bring the volume of the solution in the 50 mL centrifuge tube to 50 mL, resulting in a solution with a transglutaminase concentration of 10 wt%. After the solution has been allowed to stand at 4°C for 30 minutes, carefully transfer the supernatant to another centrifuge tube.

[0073] Add 4 g of the gelatin powder obtained in Preparation Example 1-1 and 1 g of sodium polyacrylate powder to the other centrifuge tube. Shake the tube for 30 minutes to obtain a mixture. Add 1.25 g of the mixture to a 10 mL first syringe. Add 5 mL of the transglutaminase solution to a 10 mL second syringe. Close the syringe caps on both syringes. Remove the syringe cap, connect the first and second syringes using a two-way valve, and alternately push the piston assemblies of the first and second syringes 20 to 30 times. For example, in step (a), use the piston assembly of the second syringe to push a portion of the solution into the first syringe to form a mixture, then use the piston assembly of the first syringe to push a portion of the mixture into the second syringe. Repeat step (a) approximately 7 times to prevent the mixture from clumping. In step (b), use the piston assembly of the second syringe to push all of the mixture into the first syringe, then use the piston assembly of the first syringe to push all of the mixture into the second syringe. Repeat step (b) approximately 18 times. A total of 25 alternate pushes of the piston assemblies of the third and fourth syringes are performed to obtain a pre-crosslinked hydrogel.

[0074] Use the plunger assembly of the second syringe to push the hydrogel into the silicone ice cube tray. Do not wait. Set the refrigerator temperature to -20°C and freeze the hydrogel in the refrigerator for 16 to 24 hours to obtain frozen hydrogel.

[0075] The freeze dryer was turned on, the temperature was lowered to about -20°C and the pressure was lowered to about 10 Torr, and the frozen hydrogel was freeze-dried for 72 hours. The pressure was then released and the freeze dryer was turned off to obtain freeze-dried hydrogel.

[0076] Grind the freeze-dried hydrocolloid in a grinder. Sieve the ground hydrocolloid using a 20-mesh sieve to obtain a powder with a particle size of approximately 850 μm or less. Dry and store the powder in a 50 mL centrifuge tube.

[0077] In this embodiment, a syringe is used to produce the powder or granule, but the present disclosure is not limited thereto.

[0078] Example 1. Gel

[0079] See Figures 1 to 4. Add 0.495 mL of liquid 51 (i.e., ddH2O) to the 3 mL first syringe 10, then add powder 52 (i.e., 148.9 mg of the powder obtained in Preparation Example 2 and 41.1 mg of β-tricalcium phosphate) to the 3 mL second syringe 20, use a two-way valve 30 to connect the first syringe 10 and the second syringe 20, and push the piston assembly 12 of the first syringe 10 and the piston assembly 22 of the second syringe 20 alternately for about 3 to 10 times, for example: Step (a) Use the piston assembly 12 of the first syringe 10 to push a portion of the liquid 51 into the second chamber 213 of the second syringe 20 along the longitudinal direction X to form a mixture 53, and then use the piston assembly 22 of the second syringe 20 to push along the longitudinal direction X. A portion of the mixture 53 was pushed toward X into the first cavity 113 of the first syringe 10, and step (a) was repeated approximately twice to prevent the mixture 53 from clumping. In step (b), the piston assembly 12 of the first syringe 10 was used to push all of the mixture 53 along the longitudinal direction X into the second cavity 213 of the second syringe 20. Then, the piston assembly 22 of the second syringe 20 was used to push all of the mixture 53 along the longitudinal direction X into the first cavity 113 of the first syringe 10, and step (b) was repeated approximately four times. In step (c), the second piston assembly 22 of the second syringe 20 was pushed along the longitudinal direction X to eject the mixture 53 from the opening 212 of the second syringe 20, thereby obtaining the gel of Example 1. Based on the total weight of the gel of Example 1, the gel comprised 21.7 wt% of powder and granules, 6 wt% of β-tricalcium phosphate, and 72.3 wt% of water.

[0080] Example 2. Gel carrying BMP2

[0081] The gel of Example 2 was prepared by the method of Example 1, except that 1 part of the powder and β-tricalcium phosphate was mixed with 2.4 to 2.8 parts of the BMP2 aqueous solution to obtain the moldable carrier low-dose gel carrying BMP2 of Example 2.

[0082] Comparative Example 1. Bone cement

[0083] A powder and a liquid solution were provided. The powder consisted of polymethylmethacrylate (PMMA), benzoyl peroxide, zirconium dioxide, and gentamicin sulfate. The liquid solution consisted of methyl methacrylate (MMA) and N,N-dimethyl-p-toluidine (DmpT). The powder and liquid solution were manually mixed and hydrated to form the bone cement of Comparative Example 2.

[0084] Comparative Example 2. Bone meal

[0085] β-Tricalcium phosphate (purchased from Spectrum Chemical) was used as the bone powder in Comparative Example 2.

[0086] Comparative Example 3. Gel

[0087] The gelatin of Preparation Example 1-1, sodium polyacrylate, and transglutaminase were added to deionized water to perform a cross-linking reaction, thereby obtaining the gel of Comparative Example 3.

[0088] Feature Analysis

[0089] The left image of Figure 5 shows an SEM photograph of the entire gel of Example 1. The middle and right images of Figure 5 show SEM photographs of the surface structure of the gel of Example 1. As shown in the middle image of Figure 5, the gel of the present disclosure has a porous structure. As shown in the right image of Figure 5, β-tricalcium phosphate appears as white spherical crystals in the SEM photograph, without affecting the porosity of the gel.

[0090] FIG6 is a graph showing the cytotoxicity test results of the gel of Example 1. Test samples of 25% gel extract, 50% gel extract, 75% gel extract, 100% gel extract, reagent control group, negative control group and positive control group are provided. According to the provisions of ISO-10993-5 biocompatibility, CCK-8 is used to detect cell mitochondrial activity. When the cell viability is greater than 70%, the test sample is considered to be non-cytotoxic. After detection and calculation by commercially available instruments, the reagent control group is considered to have a cell viability of 100% for comparison with other groups. The cell viability of the negative control group is close to that of the reagent control group, and the cell viability of the positive control group is extremely low. As shown in FIG6 , in terms of the experimental groups, each group of gel extracts has a cell viability greater than 70%, which is close to the cell viability of the reagent control group. It can be seen that the gel disclosed in the present invention does not produce cytotoxicity and is a biocompatible material.

[0091] Figure 7 shows the molding temperatures of Example 1 and Comparative Example 1. As shown in Figure 7, the bone cement of Comparative Example 1 reached a temperature of approximately 26°C 0.5 minutes after molding, gradually increasing in temperature to a high temperature of approximately 72°C 10 minutes after molding. In contrast, the gel of Example 1 of the present disclosure reached temperatures of approximately 29.67±0.58°C and 27.65±0.58°C only 0.5 minutes after molding, before cooling to room temperature 10 minutes after molding. Therefore, the gel of the present disclosure does not generate high temperatures, thus avoiding damage to surrounding tissues.

[0092] Application in bone defects

[0093] Figure 8 shows CT images of the gel of Example 1 and the bone cement of Comparative Example 1 used as fillers for defects in the condyles of the femur of rabbits. As shown in the left image of Figure 8 , a bone defect approximately 6 mm in diameter and 10 mm deep was drilled into the condyles of the femur of New Zealand white rabbits (Oryctolagus cuniculus). The filler was then placed into the affected area, and the soft tissue was surgically sutured. A CT image was taken eight weeks after the filler was implanted, and the results are recorded in the right image of Figure 8 . As shown in the histological images in the right image of Figure 8 , eight weeks after the filler was implanted, the blank control group still showed a defect. The bone cement of Comparative Example 1 maintained its overall structure and showed a significant difference in density from the surrounding native bone. The gel of Example 1 resulted in a bone defect with a structure similar to native cancellous bone. This demonstrates that the gel of the present disclosure can help cancellous bone heal, achieving the technical effect of bone regeneration.

[0094] In addition, at 8 weeks after the implantation of the aforementioned filler, the affected area (i.e., the implanted area) was prepared into undecalcified hard tissue sections. Specifically, the specimen was embedded in PMMA and sliced ​​to 500 μm, then ground to 50 to 80 μm. Collagen was then stained with aniline blue, and calcium was stained with silver nitrate. The results are recorded in the right figure of Figure 8. Referring to the histological section results on the right side of Figure 8 , at eight weeks after implantation, the blank group still maintained a defect, with no signs of tissue growth entering the gap. At eight weeks after implantation, the bone cement of Comparative Example 1 was merely encapsulated by surrounding tissue, but no bone tissue had grown into the cement. This indicates that the bone cement of Comparative Example 1 lacked osteoconductivity, resulting in poor osseointegration. At eight weeks after implantation, the gel of Example 1 had been replaced by tissue, with collagen, indicated by aniline blue (blue), observed in the filled area, indicating that surrounding bone tissue had begun to replace the original defect. Furthermore, calcification, indicated by silver nitrate (black), was observed in the filled area, indicating that mineralization had occurred. Therefore, the gel of the present disclosure can indeed achieve the technical effect of promoting bone defect recovery.

[0095] Figure 9A shows CT images of the gel of Example 1, the bone cement of Comparative Example 1, and the bone powder of Comparative Example 2 used as fillers for defects in porcine tibiae. An electric drill was used to drill a hole approximately 50 mm in diameter and 60 mm deep in the porcine tibiae. As shown in the upper and middle figures of Figure 9A , the drilled porcine tibiae were left unfilled as a blank control, while the drilled porcine tibiae were filled with the bone cement of Comparative Example 1, the bone powder of Comparative Example 2, and the gel of Example 1, respectively. As shown in the lower figure of Figure 9A , bone screws, serving as implants, were inserted into the holes to observe the filling effects of the fillers. The results in Figure 9A show that while the bone cement of Comparative Example 2 has filling properties, it suffers from the aforementioned problem of high polymerization temperatures damaging surrounding bone tissue. The bone powder of Comparative Example 2 also exhibits poor filling properties, dispersing as powder around the implant. The gel of Example 1, on the other hand, has good filling properties, although it contains some pores. After implantation, the gel remains between the implant and the bone tissue. Therefore, compared with bone cement and bone powder, the gel disclosed in the present invention can achieve excellent bone integration effect.

[0096] Figure 9B shows the pull-out test results for the implants of the example and control groups shown in the lower image of Figure 9A. Using commercially available instruments, pull-out tests were conducted on the control group and on bone screws filled with the gel from Example 1. The results are recorded in Figure 9B. As shown in Figure 9B, at three weeks after implantation, the gel from Example 1 exhibited a greater pull-out strength than the control group. Furthermore, at six weeks after implantation, the gel from Example 1 exhibited a pull-out strength approximately 130% greater than that of the control group. This demonstrates that the gel disclosed herein can enhance the stability of bone implants.

[0097] In addition, a universal testing machine (UTM) was used to measure the yield stress, Young's modulus, and failure stress of the gel of Example 1, the bone cement of Comparative Example 1, osteoporotic cancellous bone, and cancellous bone of healthy bones. The results are recorded in Table 1 below.

[0098] [Table 1]

[0099] As shown in Table 1, the bone cement of Comparative Example 1 exhibits a Young's modulus far higher than that of cancellous bone, while the gel of Example 1 has a Young's modulus similar to that of cancellous bone in osteoporosis patients. Due to its high Young's modulus, the bone cement of Comparative Example 1 is not helpful in helping patients recover from bone damage. In contrast, the gel of the present disclosure, due to its suitable Young's modulus and its soft and elastic properties, does not create stress shielding or cause additional damage to non-implanted areas when used with rigid implants in the cancellous bone of osteoporosis patients. Therefore, the gel of the present disclosure can replace bone cement, providing a buffer zone between the implant and native bone.

[0100] FIG10A is a confocal microscopic photograph of bone cell mineralization according to at least one embodiment of the present disclosure. Mouse osteoblasts (mOBs), a gel with a volume of 1200 μL / dose (μL / dose), and a mold with a volume of 200 μL / mold (μL / mold) were provided. A concentration of 1.2×10 6 The cells / dose of mOB were mixed with the gel of Example 1, wherein mOB and 0.5 mL of α-MEM replaced ddH2O. The mixed gel and cells were squeezed into the mold. The number of cells was calculated to be 2×10 5Cells / mold. After smoothing the gel in the mold using the bottom of the culture dish and surgical instruments, turn the mold upside down and stack 4 304 stainless steel washers on top. After stacking is completed, inject 7mL of α-MEM culture medium to submerge the mold. Samples were taken on the 0th, 3rd, 7th and 14th days of culture, and the α-MEM culture medium was replaced every 2 days. At the end of the experiment, the culture medium was removed and 7mL of 1x phosphate buffer solution (PBS) was added to wash twice. After removing 1x PBS, 7mL of 4% paraformaldehyde (PFA) was used to fix the gel for 24 hours to prepare a specimen. After the fixation time is over, add 1x PBS to wash the specimen. The specimens were then fixed with agarose to facilitate sectioning. Hoechst 33342 stain was then added to stain the nuclei, and xylenol orange (XO) was added to stain the mineralization. Mounting slides were then prepared and observed and photographed using a confocal microscope. The results are recorded in Figure 10A. As shown in Figure 10A, on day 0, the number of nuclei in the gel of Example 1 was minimal, and the gel exhibited a sharp XO signal due to its inclusion of β-tricalcium phosphate. On day 3, the number of nuclei in the gel increased, and the XO signal at the nuclei was above background, indicating that the cells were beginning to secrete mineralization. On day 7, the number of nuclei in the gel continued to increase, and mineralization signals were also observed. The number of nuclei in the gel continued to increase until day 14, and the mineralization signal was still abundant. The cell nuclei marked by Hoechst 33342 signals were integrated, and the proliferation performance was described by magnification with day 0 as the benchmark, and the results were recorded in Figure 10B. As shown in Figure 10B, the proliferation magnification of the cell nuclei in the gel of Example 1 was about 2.6±1.13 times on day 3, about 14.9±2.07 times on day 7, and about 15.6±2.13 times on day 14. Compared with the number of cell nuclei on day 0, the number of cell nuclei on day 14 was significantly different (P-value < 0.0001). The above results show that in the gel of the present disclosure, osteoblasts (OB) can normally secrete mineralized substances, and the Ca in the gel is 2+ The concentration is beneficial to the proliferation and survival of OB cells.

[0101] Drug carrying capacity

[0102] Figure 11 shows the drug release curve of the gel in Example 2. As shown in Figure 11, the gel in Example 2 continuously released BMP2 for seven days, with 36.4% of BMP2 released within 24 hours and 34.1% released within 48 hours. Because BMP2 release peaked within 24 hours, bone cell mineralization testing was conducted again on the 24-hour extracts from the gels in Examples 1 and 2.

[0103] FIG12A is a confocal microscopic photograph showing the mineralization of bone cells in the gels of Examples 1 and 2 of the present disclosure. Only xylenol orange (XO) was added to the 24-hour extracts of the gels of Example 1 and Example 2, and the results of bone cell mineralization on day 0 were observed and photographed using a confocal microscope, and the results are recorded in FIG12A . As shown in FIG12A , the bright field, xylenol orange, and merged photographs of the control group did not show the formation of minerals in bone cells; the bright field, xylenol orange, and merged photographs of Example 1 showed the formation of minerals in bone cells; and the bright field, xylenol orange, and merged photographs of Example 2 showed more minerals formed in bone cells. FIG12B is a histogram showing the amount of bone cell mineralization in the gels of Examples 1 and 2 of the present disclosure. As shown in Figure 12B, the osteoblasts treated with the control group had a mineralization level of only approximately 0.01, the osteoblasts treated with the gel of Example 1 had a mineralization level of approximately 0.02, and the osteoblasts treated with the 24-hour extract of the gel of Example 2 had a mineralization level of approximately 0.05 to 0.06. These results confirm that the 24-hour extracts of the gel of Example 1 and the gel of Example 2 help promote osteoblast mineralization, and that the gel of Example 2 has a significantly different (P-value < 0.05) amount of osteoblast mineralization compared to the gel of Example 1 due to its inclusion of BMP2.

[0104] Figure 13 shows CT images of bone defect healing in mice 29 days after surgery. Three groups of mouse bone defect models were provided, one group served as the control group, and the other two groups were administered with the gel of Example 1 and the gel of Example 2, respectively. The mice were sacrificed 29 days after surgery, and the healing of the fractured bones in each group of mice was observed through micro-computed tomography images. As shown in the upper left and lower left figures of Figure 13, the fractured bones in the control group did not heal. As shown in the upper middle and lower middle figures of Figure 13, the fractured bones in which the gel of Example 1 was administered healed. As shown in the upper right and lower right figures of Figure 13, the fractured bones in which the gel of Example 2 was administered healed. Therefore, both the gel of Example 1 and the gel of Example 2 have the effect of bone repair.

[0105] Figure 14 (top) shows a schematic diagram of the gel structure in Comparative Example 3, while Figure 14 (bottom) shows a schematic diagram of the gel structure in Example 1. As shown in Figure 14 (top), after the gelatin, transglutaminase, and sodium polyacrylate, which have not been pre-crosslinked, undergo a hydration reaction to form a gel, sodium polyacrylate is highly absorbent (typically absorbing 200 to 300 times its mass in water). This results in a significant volume expansion of the gel, for example, but not limited to, over 150 times. Therefore, the gel volume in Comparative Example 3 is uncontrollable. When used to fill bone defects or as a filler between a bone defect and an implant, the gel can absorb a large amount of water from the surrounding tissue, causing excessive swelling. This can lead to adverse effects such as swelling or pain in the affected area, excessive gel overflow outside the affected area, or implant loss. As shown in the lower figure of Figure 14 , after the pre-crosslinked gelatin, transglutaminase, and sodium polyacrylate undergo a hydration reaction to form a gel, the crosslinked gelatin and transglutaminase limit the overall volume expansion of the gel to a small extent, while the sodium polyacrylate still imparts a certain degree of hydrophilicity to the gel. Because Example 1 is a reaction product with controllable gel volume, when used to fill bone defects or as a filler between a bone defect and an implant, the limited swelling allows only slight expansion into the gaps in native cancellous bone and / or the surface gaps of an implant (such as, but not limited to, a bone screw or bone plate), thereby securing the implant and / or facilitating cell regeneration. Excessive swelling, which could cause swelling or pain in the affected area, or excessive overflow beyond the affected area, is avoided.

[0106] Figure 15 (top) shows an SEM photograph of the gel in Example 1, and Figure 15 (bottom) shows an SEM photograph of the gel in Comparative Example 3. As shown in Figure 15 (top), gelatin, transglutaminase, and sodium polyacrylate are pre-crosslinked to form a porous powder. When the gel is formed at a fixed volume of aqueous solution, the aqueous solution quickly and evenly diffuses into the pores of the porous structure of the powder. Furthermore, the gel does not absorb water from the body, causing excessive swelling. As shown in Figure 15 (bottom), gelatin, transglutaminase, and sodium polyacrylate are not pre-crosslinked to form a porous structure. When the gel is formed at a fixed volume of aqueous solution, the gel absorbs a large amount of water from the body, causing excessive swelling.

[0107] Figure 16 (top) shows an SEM photograph of the gelatin prepared in Preparation Example 1-2, while Figure 16 (bottom) shows an SEM photograph of the gelatin prepared in Preparation Example 1-1. As shown in Figure 16 (top), gelatin that had not been reconstituted, freeze-dried, and ground into powder appeared granular. As shown in Figure 16 (bottom), gelatin that had been reconstituted, freeze-dried, and ground into powder appeared flake-like. Its larger surface area makes it easier to reconstitute than granular gelatin powder.

[0108] Figure 17 shows an SEM photograph of the powder of Preparation Example 2. As shown in Figure 17 , the powder of the present disclosure has a sponge-like structure comprising a plurality of interconnected pores.

[0109] In summary, the gel disclosed herein can be applied to, for example: patients with osteoporosis who need permanent bone screws, elderly people who need permanent bone screws, bone defect sites that need to avoid poor bone integration between implants and surrounding natural bones, bone defect sites that need to induce or fill stress-free bone tissue and promote its regeneration, or patients who need to fix dental implant screws, but the disclosure is not limited to these. The above are only some embodiments of the disclosure, and those with ordinary knowledge in the relevant field will be able to easily understand that various modifications and changes can be made to each embodiment without departing from the teachings of the disclosure. Therefore, all equivalent changes and modifications made in accordance with the scope of the patent application of the present disclosure should fall within the scope of the disclosure. [Explanation of Symbols]

[0110] 10: First syringe 113: First cavity 12: First piston assembly 20: Second syringe 212: Opening 213: Second cavity 22: Second piston assembly 30: Connector 51: Liquid 52: Powder 53: Mixture X: Long axis direction

Claims

1. A gel comprising: A powder comprising a reaction product of transglutaminase, gelatin, and sodium polyacrylate, wherein the transglutaminase is covalently cross-linked to the gelatin, and the transglutaminase is covalently cross-linked to the sodium polyacrylate; beta-tricalcium phosphate; and carrier.

2. The gel according to claim 1, wherein The total weight of the gel comprises: 10 to 30 wt% of the powder; and 2 to 10 wt% of the β-tricalcium phosphate.

3. The gel according to claim 1 or 2, wherein the gelatin is in the form of gelatin granules, and the particle size of the gelatin granules is less than 850 μm.

4. The gel according to any one of claims 1 to 3, wherein The weight ratio of the transglutaminase, the gelatin and the sodium polyacrylate is 4 to 6:3 to 5:

1.

5. The gel according to any one of claims 1 to 4, wherein The powder, the β-tricalcium phosphate, and the carrier interact to form a structure, wherein the transglutaminase promotes a cross-linking reaction between the gelatin and a portion of the OH groups of the sodium polyacrylate to form a covalent bond, the calcium ions of the β-tricalcium phosphate react with the carboxyl groups of the sodium polyacrylate, and another portion of the OH groups of the sodium polyacrylate forms hydrogen bonds with the carrier.

6. The gel according to any one of claims 1 to 5, wherein The reaction product has a sponge-like structure including a plurality of interconnected pores.

7. The gel according to claim 6, wherein The sponge-like structure also includes a flake-like structure.

8. The gel according to any one of claims 1 to 7, wherein The particle size of the powder is 850 μm or less.

9. The gel according to any one of claims 1 to 8, further comprising a drug and / or a cell preparation.

10. A method for producing the gel according to any one of claims 1 to 9, comprising: Providing a solution containing the transglutaminase, and a mixture of the gelatin and the sodium polyacrylate; mixing the solution and the mixture to obtain the reaction product; freeze-drying and grinding the reaction product to obtain the powder; as well as The powder, the β-tricalcium phosphate and the carrier are mixed to obtain the gel.

11. The method of claim 10, wherein: The gelatin is in the form of gelatin powder and particles.

12. The method according to claim 10 or 11, further comprising: providing a solution containing colloidal gelatin; as well as The solution containing the colloidal gelatin is freeze-dried and ground to obtain the gelatin powder.

13. The method according to any one of claims 10 to 12, wherein: The mixing of the powder, the β-tricalcium phosphate and the carrier comprises: storing the carrier in a first syringe, and storing the powder and the β-tricalcium phosphate in a second syringe; connecting the first syringe and the second syringe via a connector, thereby communicating the first syringe and the second syringe; and The piston assembly of the first syringe and the piston assembly of the second syringe are pushed back and forth simultaneously to obtain the gel.

14. The method of claim 13, wherein: When the first syringe is not connected to the second syringe, the piston assembly of the first syringe or the piston assembly of the second syringe is used to push the gel out of the first syringe or the second syringe.

15. Use of the gel according to any one of claims 1 to 9 for preparing a pharmaceutical composition for promoting tissue repair or regeneration.

16. The use according to claim 15, wherein The tissue is bone tissue.

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