A pH-responsive magnesium ion-collagen injectable in-situ crosslinking hydrogel as well as a preparation method and application thereof

Abstract

The application provides a kind of pH response magnesium ion-collagen injectable in-situ crosslinking hydrogel and its preparation method and application, belong to bone repair material technical field.The preparation raw material of the pH response magnesium ion-collagen injectable in-situ crosslinking hydrogel of the application includes type I collagen, magnesium source, polyphenol, acid solution;The mass fraction of type I collagen in acid solution is 0.3~0.8%, the concentration of magnesium ion in magnesium source in acid solution is 80~120mmol / L;The mass ratio of magnesium source and polyphenol is 2~4:3~6.The acid environment in the acid collagen solution of the application destroys the hydrogen bond between type I collagen molecules, so that collagen monomers are dispersed and stably exist;It can keep liquid state for a long time, and is suitable for injection;After magnesium ion-collagen composite precursor solution enters physiological weak alkaline pH environment in vivo, the charge state of collagen molecules changes, magnesium ion and collagen molecules carboxyl, amino occur ion coordination dynamic crosslinking, and spontaneously solidify into shape.

Description

Technical Field

[0001] This invention relates to the field of bone repair materials technology, and in particular to a pH-responsive magnesium ion-collagen injectable in-situ crosslinked hydrogel, its preparation method, and its application. Background Technology

[0002] Osteoporotic bone defects and nonunion fractures in the elderly are common skeletal diseases in aging societies. Currently available bone repair materials are mostly prefabricated solid scaffolds, which require surgical incision and implantation, resulting in significant trauma and inability to adapt to irregular bone defect morphologies.

[0003] Hydrogels, due to their locally soft, moist surface and biomimetic structure, can significantly avoid irritation to biological tissues, thus improving biocompatibility. Injectable hydrogels, with their unique physicochemical properties, such as unique hydrophilicity, water retention, and biomimetic characteristics, good biocompatibility, special hydrodynamic properties, and multiple environmental responsiveness, have been widely used in drug delivery and tissue repair engineering. Collagen artificial bone materials possess components and structures similar to natural bone, good biocompatibility and bioactivity, and biodegradability that better matches new bone formation. They can effectively improve problems such as infection, immune rejection, and slow in vivo degradation of ceramic biomaterials in bone transplantation, making them one of the most promising biomaterials in the field of bone repair. Traditional collagen hydrogels often use chemical cross-linking agents, which have problems such as cytotoxicity and uncontrollable degradation.

[0004] Mg 2+ It can significantly enhance osteoblast adhesion and diffusion, promoting the mineralization process; it can effectively promote osteogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs) and promote in situ bone regeneration. Most existing collagen-magnesium composite systems are directly blended and molded, without utilizing the design concept of acidic pre-dissolution and in situ cross-linking triggered under physiological pH conditions in vivo, making it difficult to meet the three core requirements of room temperature injection, no premature coagulation in vitro, and adaptive molding in vivo.

[0005] To address the aforementioned technological gaps, there is an urgent need to provide a novel, chemical-free, pH-responsive, injectable magnesium ion-collagen bone repair crosslinking hydrogel. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing bone repair materials, such as large trauma, poor adaptability, need for chemical cross-linking, and insufficient controllability in molding, by providing a pH-responsive magnesium ion-collagen injectable in-situ cross-linked hydrogel, its preparation method, and its application.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a pH-responsive magnesium ion-collagen injectable in situ crosslinked hydrogel, wherein the raw materials for preparing the pH-responsive magnesium ion-collagen injectable in situ crosslinked hydrogel include type I collagen, magnesium source, polyphenols, and acid solution; The mass fraction of type I collagen in the acid solution is 0.3-0.8%, the concentration of magnesium ions in the magnesium source in the acid solution is 80-120 mmol / L, and the mass ratio of magnesium source to polyphenol is 2-4:3-6.

[0008] Preferably, the type I collagen is one or more of mouse-derived collagen, bovine-derived collagen, and porcine-derived collagen.

[0009] Preferably, the magnesium source comprises one or more of magnesium chloride, magnesium nitrate, magnesium acetate, and magnesium bisulfate.

[0010] Preferably, the polyphenols comprise epigallocatechin gallate and / or quercetin.

[0011] Preferably, the acid solution is an aqueous solution of glacial acetic acid and / or an aqueous solution of phosphoric acid, and the concentration of the acid solution is 0.03~0.07 mol / L.

[0012] The present invention also provides a method for preparing the pH-responsive magnesium ion-collagen injectable in-situ crosslinked hydrogel, comprising the following steps: 1) Dissolve type I collagen in an acidic solution to obtain an acidic collagen solution; 2) Mix the magnesium source, polyphenols and acidic collagen solution to obtain a magnesium ion-collagen composite precursor solution; 3) The magnesium ion-collagen composite precursor solution is cross-linked in situ under weakly alkaline conditions to obtain a pH-responsive magnesium ion-collagen injectable in situ cross-linked hydrogel.

[0013] Preferably, in step 1), the type I collagen is dissolved in the acid solution under stirring conditions, and in step 2), the mixing is carried out under stirring conditions, with the stirring rate being 80~300 rpm.

[0014] The present invention also provides the application of the pH-responsive magnesium ion-collagen injectable in situ crosslinked hydrogel in the preparation of bone repair materials.

[0015] The beneficial effects of this invention are: 1) In the acidic collagen solution of the present invention, the acidic environment disrupts the hydrogen bonds between type I collagen molecules, allowing collagen monomolecules to disperse and exist stably. Under acidic conditions, the magnesium ions in the magnesium ion-collagen composite precursor solution only physically mix with collagen without chemical cross-linking, and can remain in a liquid state for a long time, making it suitable for injection. After the magnesium ion-collagen composite precursor solution enters the physiologically weakly alkaline pH environment in the body, the charge state of collagen molecules changes, and magnesium ions undergo ionic coordination dynamic cross-linking with the carboxyl and amino groups of collagen molecules, without the need for external chemical cross-linking agents, and spontaneously solidify and form.

[0016] 2) In this invention, collagen is dissolved in an acid solution at room temperature, and then magnesium salt and polyphenols are added and stirred and mixed. No chemical cross-linking agent or pre-made mold is required throughout the process. In-situ molding is triggered by physiological pH conditions in the body.

[0017] 3) The collagen hydrogel of the present invention has good mechanical strength and mechanical properties, and maintains the osteogenic space well; the chelation of magnesium ions changes the original structure of polyphenols. Under pH response, polyphenols and magnesium ions are released. Polyphenols can absorb more reactive oxygen species, eliminate inflammation, and promote wound healing; the release of metal ions exposes more phenolic hydroxyl groups, which plays a synergistic role in antibacterial and hemostatic effects. Detailed Implementation

[0018] This invention provides a pH-responsive magnesium ion-collagen injectable in situ crosslinked hydrogel, wherein the raw materials for preparing the pH-responsive magnesium ion-collagen injectable in situ crosslinked hydrogel include type I collagen, magnesium source, polyphenols, and acid solution; The mass fraction of type I collagen in the acid solution is 0.3-0.8%, the concentration of magnesium ions in the magnesium source in the acid solution is 80-120 mmol / L, and the mass ratio of magnesium source to polyphenol is 2-4:3-6.

[0019] In this invention, the mass fraction of type I collagen in the acid solution is preferably 0.4-0.7%, more preferably 0.5-0.6%, the concentration of magnesium ions in the magnesium source in the acid solution is preferably 90-110 mmol / L, more preferably 100 mmol / L, and the mass ratio of magnesium source to polyphenol is preferably 2.5-3.5:3.5-5.5, more preferably 3:4-5.

[0020] In this invention, the type I collagen is preferably one or more of mouse-derived collagen, bovine-derived collagen, and porcine-derived collagen.

[0021] In this invention, the magnesium source preferably comprises one or more of magnesium chloride, magnesium nitrate, magnesium acetate, and magnesium bisulfate.

[0022] In this invention, magnesium ions promote stem cell proliferation and osteogenic differentiation, promote angiogenesis at the defect site, regulate nerve regeneration at the defect site, induce the expression of osteogenic-related genes, and enhance the mineralization of the extracellular matrix. When magnesium ions are implanted into materials to promote bone formation, they can, to some extent, improve the problems of poor collagen mechanical strength and premature collapse during bone healing. The addition of magnesium can provide a more suitable local environment for the bone mineralization process. Magnesium ions can not only control the assembly speed of collagen and affect its assembly structure, but also control the nucleation and crystallization process of minerals.

[0023] In this invention, the polyphenol preferably comprises epigallocatechin gallate and / or quercetin; the polyphenol contains multiple phenolic hydroxyl groups, which can chelate magnesium ions and play an antioxidant, antibacterial and hemostatic role.

[0024] In this invention, the acid solution is preferably an aqueous solution of glacial acetic acid and / or an aqueous solution of phosphoric acid, and the concentration of the acid solution is preferably 0.03~0.07 mol / L, more preferably 0.04~0.06 mol / L, and even more preferably 0.05 mol / L.

[0025] The present invention also provides a method for preparing the pH-responsive magnesium ion-collagen injectable in-situ crosslinked hydrogel, comprising the following steps: 1) Dissolve type I collagen in an acidic solution to obtain an acidic collagen solution; 2) Mix the magnesium source, polyphenols and acidic collagen solution to obtain a magnesium ion-collagen composite precursor solution; 3) The magnesium ion-collagen composite precursor solution is cross-linked in situ under weakly alkaline conditions to obtain a pH-responsive magnesium ion-collagen injectable in situ cross-linked hydrogel.

[0026] In this invention, the dissolution of type I collagen in the acid solution in step 1) is preferably carried out under stirring conditions, and the mixing in step 2) is preferably carried out under stirring conditions. The stirring rate is preferably 80~300 rpm, more preferably 120~260 rpm, and even more preferably 150~200 rpm.

[0027] In this invention, the pH value of the weakly alkaline condition in step 3) is 7.25~7.45, which is consistent with the pH value of the physiological environment in the body. The magnesium ion-collagen composite precursor solution is minimally invasively injected into the bone defect site. The pH of the physiological environment in the body spontaneously triggers the ion coordination effect, and the magnesium ions and collagen molecules undergo rapid in-situ cross-linking to form a three-dimensional porous biomimetic bone repair scaffold. The magnesium ions and polyphenols chelate to form a nano complex.

[0028] In this invention, the acidic collagen solution is a uniform and transparent acidic collagen solution with a pH value of 3.5~4.7; the magnesium ion-collagen composite precursor solution is in a uniform flow state under acidic conditions, does not undergo cross-linking, does not flocculate or precipitate, has good fluidity, and is injectable.

[0029] The present invention also provides the application of the pH-responsive magnesium ion-collagen injectable in situ crosslinked hydrogel in the preparation of bone repair materials.

[0030] The pH-responsive magnesium ion-collagen injectable in-situ crosslinked hydrogel of the present invention is a biomimetic bone repair hydrogel used for minimally invasive tissue engineering repair of osteoporotic bone defects, delayed fracture healing, and irregular bone defects.

[0031] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0032] Example 1

[0033] The raw materials for preparing the pH-responsive magnesium ion-collagen composite precursor solution in this embodiment include rat tail type I collagen, magnesium acetate, epigallocatechin gallate, and an aqueous solution of glacial acetic acid with a concentration of 0.05 mol / L. The mass fraction of rat tail type I collagen in the aqueous solution of glacial acetic acid is 0.5%, and the concentration of magnesium ions in magnesium acetate in the aqueous solution of glacial acetic acid is 100 mmol / L. The mass ratio of magnesium acetate to epigallocatechin gallate is 3:5.

[0034] Type I collagen from rat tail was dissolved in an aqueous solution of glacial acetic acid and stirred at 150 rpm to obtain a homogeneous acidic collagen solution. Magnesium acetate, epigallocatechin gallate, and the acidic collagen solution were mixed and stirred at 150 rpm to obtain a homogeneous magnesium ion-collagen complex precursor solution.

[0035] Example 2

[0036] The raw materials for preparing the pH-responsive magnesium ion-collagen composite precursor solution in this embodiment include bovine type I collagen, magnesium chloride, quercetin, and a 0.04 mol / L aqueous solution of phosphoric acid; the mass fraction of bovine type I collagen in the aqueous solution of phosphoric acid is 0.7%, the concentration of magnesium ions in magnesium chloride in the aqueous solution of phosphoric acid is 120 mmol / L, and the mass ratio of magnesium chloride to quercetin is 2:3.

[0037] Type I bovine collagen was dissolved in an aqueous phosphoric acid solution and stirred at 200 rpm to obtain a homogeneous acidic collagen solution. Magnesium chloride, quercetin, and the acidic collagen solution were then stirred and mixed at 200 rpm to obtain a homogeneous magnesium ion-collagen complex precursor solution.

[0038] Example 3

[0039] The raw materials for preparing the pH-responsive magnesium ion-collagen composite precursor solution in this embodiment include rat tail type I collagen, magnesium nitrate, epigallocatechin gallate, and an aqueous solution of glacial acetic acid with a concentration of 0.06 mol / L. The mass fraction of rat tail type I collagen in the aqueous solution of glacial acetic acid is 0.4%, and the concentration of magnesium ions in the aqueous solution of glacial acetic acid is 90 mmol / L. The mass ratio of magnesium nitrate to epigallocatechin gallate is 4:5.

[0040] Type I collagen from rat tail was dissolved in an aqueous solution of glacial acetic acid and stirred at 220 rpm to obtain a homogeneous acidic collagen solution. Magnesium nitrate, epigallocatechin gallate, and the acidic collagen solution were mixed and stirred at 220 rpm to obtain a homogeneous magnesium ion-collagen complex precursor solution.

[0041] The performance of the magnesium ion-collagen composite precursor liquid and the solidified hydrogel of Examples 1-3 was tested.

[0042] (1) The magnesium ion-collagen composite precursor solution of Examples 1 to 3 of the present invention is an acidic solution (pH value of 3.5 to 4.7). Under acidic conditions, magnesium ions, polyphenols and collagen are physically mixed without chemical cross-linking and chelation, maintaining a liquid state and having good injectability.

[0043] (2) Biocompatibility: MC3T3-E1 cells were cultured at a concentration of 3×10⁻⁶. 3 The cells were seeded at a density of 100 μL in the magnesium ion-collagen composite precursor solutions of Examples 1-3, cultured for 7 days, and cell morphology was observed. Live and dead cell staining was performed on MC3T3-E1 cells, and normal cell morphology was observed with very few dead cells. The precursor solution and hydrogel of the present invention have good biocompatibility, which is conducive to the proliferation and growth of MC3T3-E1 cells.

[0044] (3) Promote cell growth and bone repair: Prepare a pH solution with the same pH as the physiological pH in the human body using 0.05 mol / L acetic acid solution (pH values ​​of acetic acid solution are 7.25, 7.35 and 7.45 respectively). Place the magnesium ion-collagen composite precursor solution of Examples 1 to 3 into the above acetic acid solution at 37°C. Magnesium ions undergo dynamic cross-linking with carboxyl and amino groups of collagen molecules through ion coordination. Hydrogel is formed in situ by pH triggering. Magnesium ions and polyphenols chelate to form nano-complexes loaded on the hydrogel.

[0045] The hydrogels from Examples 1-3 were immersed in α-MEM medium supplemented with 10% bovine serum and 1% penicillin and streptomycin (0.5% each), and incubated at 37°C for 4 hours. The medium was then replaced, and the mixture was co-cultured at 37°C for 7 days. During co-culture, the medium was replaced every day with α-MEM medium supplemented with 10% serum and 1% penicillin and streptomycin. After 7 days of co-culture, the samples were removed and implanted subcutaneously into nude mice. The materials were then removed, paraffin-embedded, and sectioned. CD31 immunofluorescence staining was performed, and the sections were observed under a laser confocal microscope. CD31 immunofluorescence staining revealed a large number of new blood vessels in the hydrogels of Examples 1-3, indicating that the hydrogels of this invention can support stem cell growth, induce angiogenesis, and promote new bone formation.

[0046] The magnesium ion-collagen composite precursor solution of the present invention is in liquid state and has good injectability. After entering the physiological weakly alkaline pH environment in the body (pH value of 7.25~7.45), it can spontaneously solidify to form a three-dimensional porous biomimetic bone repair scaffold, which can be used for minimally invasive tissue engineering repair of osteoporotic bone defects, delayed fracture healing, and irregular bone defects.

[0047] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A pH-responsive magnesium ion-collagen injectable in-situ crosslinked hydrogel, characterized in that, The raw materials for preparing the pH-responsive magnesium ion-collagen injectable in situ crosslinked hydrogel include type I collagen, magnesium source, polyphenols, and acid solution. The mass fraction of type I collagen in the acid solution is 0.3-0.8%, the concentration of magnesium ions in the magnesium source in the acid solution is 80-120 mmol / L, and the mass ratio of magnesium source to polyphenol is 2-4:3-6.

2. The pH-responsive magnesium ion-collagen injectable in-situ crosslinked hydrogel according to claim 1, characterized in that, The type I collagen is one or more of mouse-derived collagen, bovine-derived collagen, and porcine-derived collagen.

3. The pH-responsive magnesium ion-collagen injectable in-situ crosslinked hydrogel according to claim 1 or 2, characterized in that, The magnesium source comprises one or more of magnesium chloride, magnesium nitrate, magnesium acetate, and magnesium bisulfate.

4. The pH-responsive magnesium ion-collagen injectable in-situ crosslinked hydrogel according to claim 3, characterized in that, The polyphenols comprise epigallocatechin gallate and / or quercetin.

5. The pH-responsive magnesium ion-collagen injectable in-situ crosslinked hydrogel according to claim 3, characterized in that, The acid solution is an aqueous solution of glacial acetic acid and / or an aqueous solution of phosphoric acid, with a concentration of 0.03~0.07 mol / L.

6. The method for preparing the pH-responsive magnesium ion-collagen injectable in-situ crosslinked hydrogel according to any one of claims 1 to 5, characterized in that, It includes the following steps: 1) Dissolve type I collagen in an acidic solution to obtain an acidic collagen solution; 2) Mix the magnesium source, polyphenols and acidic collagen solution to obtain a magnesium ion-collagen composite precursor solution; 3) The magnesium ion-collagen composite precursor solution is cross-linked in situ under weakly alkaline conditions to obtain a pH-responsive magnesium ion-collagen injectable in situ cross-linked hydrogel.

7. The preparation method according to claim 6, characterized in that, Step 1) The type I collagen is dissolved in the acid solution under stirring conditions. Step 2) The mixing is carried out under stirring conditions, and the stirring speed is 80~300 rpm.

8. The application of the pH-responsive magnesium ion-collagen injectable in-situ crosslinked hydrogel according to any one of claims 1 to 5 in the preparation of bone repair materials.