WO2021031726A1 - 一种可注射原位生孔水凝胶体系及其制备方法和用途 - Google Patents

一种可注射原位生孔水凝胶体系及其制备方法和用途 Download PDF

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WO2021031726A1
WO2021031726A1 PCT/CN2020/100635 CN2020100635W WO2021031726A1 WO 2021031726 A1 WO2021031726 A1 WO 2021031726A1 CN 2020100635 W CN2020100635 W CN 2020100635W WO 2021031726 A1 WO2021031726 A1 WO 2021031726A1
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injectable
hydrogel
metal particles
magnesium metal
situ pore
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PCT/CN2020/100635
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English (en)
French (fr)
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蒋欣泉
张文杰
唐艳梅
林思涵
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上海交通大学医学院附属第九人民医院
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Priority to US17/636,900 priority Critical patent/US11771805B2/en
Publication of WO2021031726A1 publication Critical patent/WO2021031726A1/zh

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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/02—Inorganic materials
    • A61L27/04—Metals or alloys
    • A61L27/047—Other specific metals or alloys not covered by A61L27/042 - A61L27/045 or A61L27/06
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
    • A—HUMAN NECESSITIES
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/222—Gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • A61L27/446—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with other specific inorganic fillers other than those covered by A61L27/443 or A61L27/46
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
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    • C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
    • C08J9/26—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof by elimination of a solid phase from a macromolecular composition or article, e.g. leaching out
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/06—Flowable or injectable implant compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2389/00—Characterised by the use of proteins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2405/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2401/00 or C08J2403/00
    • C08J2405/04—Alginic acid; Derivatives thereof

Definitions

  • the invention relates to the field of hydrogels, in particular to a biologically active hydrogel and a preparation method thereof.
  • Hydrogel has been widely used in the fields of tissue engineering and regenerative medicine due to its bionic structure highly similar to natural extracellular matrix, and can be used as a three-dimensional living cell culture and delivery system. Compared with traditional tissue engineering scaffold materials, the injectable hydrogel system can transport stem cells to the defect area under minimally invasive conditions, and the operation is simple and efficient. However, studies have reported that only 1-20% of the stem cells implanted into the defect area with the hydrogel can survive in the host. This is due to the fact that the hydrogel’s polymer polymer network structure retains a large amount of water and does not have an interconnected porous structure. It is not conducive to the rapid diffusion of nutrients and the growth of peripheral blood vessels, which greatly affects the biological activity and survival status of the cells loaded therein, and becomes a major obstacle to expanding the application of hydrogel tissue regeneration.
  • Hydrogels with porous structures can be prepared by traditional template methods, freeze-drying methods, phase separation methods, etc., but the gels prepared by these methods are all preformed scaffolds, losing the injectability of the gel. , Which limits the scope of its application. At the same time, some methods have harsh conditions and complex processes, which are not good for living cells and cannot make holes while carrying cells.
  • Chinese patent CN103237565B discloses a method of using oxidized alginate microspheres as a pore-forming agent, and the hydrogel containing living cells and pore-forming microspheres degrades after being injected into the body, leaving behind the gel Cavitation pores.
  • This "in-situ pore formation” method allows the gel to directly generate a pore structure in the injection zone, so that the gel has both injectability and porous structure.
  • the degradation of the microspheres in this method relies on long-term interstitial fluid penetration and contact. Under time-limited conditions, the microspheres in the gel will degrade unevenly and the pores will not be connected, which is not conducive to the survival of deep cells in the gel and rapid blood vessels. It is also found in the report that the porous gel is not effective in promoting bone regeneration. In addition, there are currently no other methods to prepare porous structures in injectable hydrogels that contain living cells.
  • the purpose of the present invention is to provide an injectable in-situ pore hydrogel system and its preparation method and application, so as to solve the problems in the prior art.
  • the present invention provides an injectable in-situ pore hydrogel system.
  • the injectable in-situ pore hydrogel system uses the injectable hydrogel as a continuous substrate phase, and separate living cells and magnesium metal particles are distributed in a continuous In the base phase, the injectable hydrogel is a precursor or prepolymer of the hydrogel, which can form a hydrogel after cross-linking.
  • the injectable hydrogel is selected from alginate, gelatin, agarose, chitosan, collagen, silk fibroin, cellulose, glucose, hyaluronic acid, chondroitin sulfate, matrigel, One or more hydrogel precursors or prepolymers of polyvinyl alcohol, polyethylene glycol and their derivatives.
  • the isolated cells are one or more combinations selected from mesenchymal stem cells, embryonic stem cells, induced multifunctional cells or adult cells.
  • the separated living cells can be obtained by tissue block attachment method, mechanical separation method, digestion separation method, suspension culture method, flow cell sorting method, and immunomagnetic bead sorting method.
  • the cell concentration in the injectable hydrogel system is 10 6 -10 7 cells/mL.
  • the average particle size of the magnesium metal particles is 20 ⁇ m-100 ⁇ m.
  • the maximum particle size is 150 ⁇ m.
  • the magnesium metal particles include flake particles and spherical particles obtained by mechanical grinding, atomization, reduction, electrolysis, and the like.
  • the mass fraction of magnesium in the magnesium metal particles exceeds 99%.
  • the mass fraction of magnesium in the magnesium metal particles exceeds 99.98%.
  • the addition amount of the magnesium metal particles in the injectable hydrogel system is 0.2 mg/mL to 2.0 mg/mL.
  • the invention also discloses a method for preparing the injectable in-situ pore hydrogel system as described above, mixing the injectable hydrogel, separated living cells and magnesium metal particles.
  • the invention also discloses the use of the injectable in-situ pore hydrogel system as described above in the preparation of tissue regeneration and repair agents.
  • the injectable in-situ pore hydrogel system when in use, is cross-linked at the tissue regeneration and repair site to form a porous hydrogel.
  • the method for linking the hydrogel is selected from one of chemical crosslinking, physical crosslinking, and chemical and physical hybrid crosslinking.
  • the crosslinking is one or more of ion crosslinking, temperature control, acid-base reaction, photoinitiation and polymerization reaction.
  • ionic crosslinking is to use an aqueous solution containing one of calcium ions, zinc ions, strontium ions, ferric ions, tripolyphosphate ions, sulfate ions, and citrate ions as the crosslinking agent; temperature control means Use 4 ⁇ 25°C deionized water bath for cooling or 37°C deionized water bath for heating; acid-base reactions use hydrochloric acid or sodium hydroxide aqueous solution as crosslinking agent; photoinitiation is use of ultraviolet light with a wavelength greater than 300nm to initiate crosslinking reaction, Or simultaneously with 1-[4-(2-hydroxyethoxy)-phenyl]-2-hydroxy-2 methyl acetone (Irgacure 2959) or 2,2-dimethoxy-2-phenylacetophenone (Irgacure 651) as a
  • the crosslinking time is 10s-30min. This cross-linking time is exactly the same as the operable time of the tissue to be repaired, so that the injectable in-situ porous hydrogel system in this application can be used as a repair agent for these tissue sites.
  • the elastic modulus is 0.1 kPa-100 kPa, and the water content exceeds 80 wt%.
  • the average diameter of the pore structure formed after the injectable in-situ pore hydrogel system is cross-linked is 100 ⁇ m to 300 ⁇ m and/or the maximum diameter is 500 ⁇ m.
  • the porosity of the porous hydrogel is not less than 50%. Preferably it is 50% to 80%.
  • the above technical solution of the present invention solves the difficulty of preparing porous structure in the injectable hydrogel encapsulating living cells in the prior art. It is proposed to add magnesium metal particles to the hydrogel, and use the magnesium metal particles to degrade in water. Hydrogen foams to make pores. The gas is formed around the magnesium metal particles, gradually expands to contact and fuse, and finally escapes through the restriction of the gel, leaving a pore structure in the gel that is interconnected and communicated with the outside world.
  • the method is feasible and universal in generally high water-containing hydrogels, has a mild reaction, does not require additional conditions, and is friendly to living cells.
  • the magnesium ions generated by the degradation of magnesium metal particles have the functions of promoting cell proliferation and differentiation, and promoting osteogenic angiogenesis, so that the hydrogel has biological activity at the same time.
  • the porous injectable hydrogel system formed by the pores of magnesium metal particles of the present invention has a porous structure, which is conducive to the rapid penetration of oxygen and nutrients, thereby facilitating the growth of surrounding vascular tissues, and effectively improving the entrapment in the gel
  • the survival rate of cells provides a new technical solution to solve the problem of low survival rate in cells in the research of regeneration and repair.
  • the pore preparation method of the present invention is simple, the steps are concise, no toxic substances are introduced, and it is friendly to living cells.
  • the present invention combines the injectability of the hydrogel with the porous structure, which is of great significance for the repair of lacunar, difficult operation, and irregular defect tissue; at the same time, the magnesium metal particles generate magnesium ions after gas generation and degradation, which can be Improve the biological activity of the gel, induce osteogenic differentiation of stem cells, promote new bone formation, and have application value in bone defect repair.
  • Figure 1 is a stereomicroscope and X-ray film characterizing the effect of porous structure of magnesium metal particles in a variety of hydrogels, and a stereomicroscope magnification is attached to observe the pore penetration.
  • A is type I collagen, which is gelatinized by acid-base reaction; B is gelatin, which is gelatinized by cooling; C is silk fibroin, which is cross-linked by polymerization; D is sodium alginate (alizarin red stain), which is Calcium ion cross-linking.
  • Figure 2 shows the pore preparation of different specifications and different dosages of magnesium metal particles in the gelatin-sodium alginate injectable hydrogel system.
  • A is the MicroCT characterization effect diagram of the pore-making situation of cutting magnesium metal particles with a particle size of 100 ⁇ m and atomized spherical magnesium metal particles with a particle size of 20 ⁇ m, 50 ⁇ m and 100 ⁇ m at the same dosage;
  • B is the addition of different magnesium metal particles under the same particle size conditions Scanning electron microscopy characterization effect diagram of the hole making of the amount (0, 0.2, 1.0, 2.0 mg/mL).
  • Fig. 3 shows the results of research on the porous hydrogel obtained by pore-making magnesium metal particles that contains living cells and improves their survival rate.
  • A is the in vitro culture of 3T3-L1 cells coated with gelatin-sodium alginate gel, and live-and-death double staining is used to detect the effect of pore preparation of magnesium metal particles on the survival rate of cells in vitro;
  • B is the gelatin-sodium alginate gel-encapsulated Luciferase And GFP double-labeled cells were implanted subcutaneously in nude mice, and in vivo fluorescence imaging was used to evaluate the effect of magnesium metal particles on the survival rate of cells in vivo.
  • Figure 4 shows the experimental effect of the hydrogel system made of magnesium metal particles in favor of peripheral blood vessel and tissue ingrowth.
  • Microfil perfusion MicroCT scan to evaluate the number of blood vessel ingrowth
  • Figure 5 shows the experimental effect of the hydrogel system made of magnesium metal particles in favor of vascularized bone regeneration and repair.
  • Sodium alginate-gelatin gel encapsulated rat bone marrow mesenchymal stem cells to repair rat distal femoral defects, evaluated by MicroCT Osteogenesis.
  • Figure 6 is an experimental effect diagram of magnesium metal particles in the hydrogel system containing living cells, which is beneficial to the repair of bone defects.
  • Sodium alginate-gelatin gel is added with magnesium metal particles, and the rats are simultaneously encapsulated or not.
  • Bone marrow-derived mesenchymal stem cells repaired rat tibia defect, MicroCT and HE staining of sections to evaluate the bone formation.
  • Step 1 Use magnesium metal particles as pore former to prepare porous injectable hydrogel
  • rat tail collagen type I 3mg / mL collagen 300 ⁇ L, 10xPBS 50 ⁇ L, dH 2 O 81 ⁇ L, 0.1mol / L NaOH solution, 69 l, was added magnesium metal particles are mixed, allowed to stand at room temperature to a gum.
  • the 15% (w/v) gelatin solution water bath is heated to 37°C, magnesium metal particles are added and mixed, and the temperature is reduced to room temperature 25°C to form a gel.
  • Magnesium metal particles can form a multi-layered pore structure in the gelatin-sodium alginate gel, and the pores are interconnected.
  • Step 2 Optimize the parameters of using magnesium metal particles as pore formers
  • the milled flake magnesium metal particles with a particle size of 100 ⁇ m and the atomized spherical magnesium metal particles with a particle size of 20 ⁇ m, 50 ⁇ m, and 100 ⁇ m were added to the gelatin-sodium alginate mixed solution described in step one in the same amount.
  • the forming mold and the gel forming method are the same step one. MicroCT scan reconstruction after gel demolding.
  • Atomized spherical magnesium metal particles with a particle size of 20 ⁇ m were added to the gelatin-sodium alginate mixture described in step 1 at the content of 0.2, 1.0, 2.0 mg/mL.
  • the control group did not add magnesium metal particles, and the forming mold and gel forming method were the same step one. After the gel was demolded, it was placed in liquid nitrogen for rapid freezing for 30 minutes. After taking it out, it was vacuumed in a freeze dryer for 4 hours to obtain a dried sample and sprayed with gold. The sample was characterized by a field emission scanning electron microscope, scanning 50 times and 100 times the field of view.
  • a porous hydrogel using magnesium metal particles to make pores improves the survival rate of cells.
  • Step 1 Use magnesium metal particles as a pore-forming agent to prepare porous hydrogels to improve cell survival in vitro
  • Step 2 Use magnesium metal particles as pore-forming agent to prepare porous injectable hydrogel to improve the survival rate of cells in vivo
  • the 15% gelatin solution, 4% sodium alginate solution and the above cell suspension were mixed uniformly in a volume ratio of 3:2:1, the final cell concentration was 10 6 -10 7 /mL, the experimental group was mixed with magnesium metal particles, and the control group had no Magnesium metal particles, 0.1mol/L CaCl 2 solution for cross-linking, 150 ⁇ L volume per sample.
  • the above-mentioned cell-containing gel sample was implanted subcutaneously on the back of nude mice. At 3 and 10 days, each nude mouse was injected intraperitoneally with fluorescein potassium salt at a dose of 150 mg/kg. After 15 minutes, the nude mice were injected anesthetized and the fluorescence intensity was analyzed by live imaging.
  • the porous injectable hydrogel made of magnesium metal particles promotes the growth of blood vessels and tissues.
  • the gel was injected into an 8-week-old male SD Subcutaneously on the back of the rat, 200 ⁇ L of each sample was injected and cross-linked with 0.1 mol/L CaCl 2 solution after injection, and washed with physiological saline three times. One week later, the descending aorta was cannulated with Microfil vascular perfusion. Take material, fix, MicroCT scan, 3D reconstruction, analyze the number of blood vessels. After the sample was scanned, it was embedded in paraffin, sectioned, stained with HE, and analyzed the amount of tissue growth.
  • Porous hydrogels made of magnesium metal particles are used to encapsulate stem cells to promote vascularized bone regeneration.
  • Step 1 Isolation and culture of rat bone marrow stem cells (rBMSCs)
  • 4-week-old SD rats were sacrificed by detachment of their necks.
  • the bilateral femurs and tibias were separated under aseptic conditions, the bilateral metaphyses were cut off, and the bone marrow cavity was washed repeatedly with DMEM medium.
  • the washing solution was collected and centrifuged at 1000 rpm for 15 minutes at room temperature and discarded. Clear, resuspend the cells, and then plate them for culture.
  • Step 3 Preparation and repair of bone defect model
  • Eight-week-old male SD rats were anesthetized to create a cylindrical defect of 2.5 mm diameter and 3 mm depth on the inner side of the distal femur.
  • the experimental group added magnesium metal particles to the gel-cell mixture, and the control group had no magnesium metal particles.
  • the defect was injected with a gel-cell mixture of 30 ⁇ L each, and after injection, a 0.1mol/L CaCl 2 solution was injected for cross-linking, washed with normal saline for 3 times, and a blank control group was set.
  • the rats were sacrificed, materials were taken, fixed, and 3D reconstruction of MicroCT scan was used to analyze the new bone condition.
  • Magnesium metal particles are used to create pores in situ in the hydrogel containing stem cells to improve the effect of defect repair.
  • Step 1 Isolation and culture of rat bone marrow stem cells (rBMSCs)
  • 4-week-old SD rats were sacrificed by detachment of their necks.
  • the bilateral femurs and tibias were separated under aseptic conditions, the bilateral metaphyses were cut off, and the bone marrow cavity was washed repeatedly with DMEM medium.
  • the washing solution was collected and centrifuged at 1000 rpm for 15 minutes at room temperature and discarded. Clear, resuspend the cells, and then plate them for culture.
  • Step 2 Preparation of injectable gel containing magnesium metal particles
  • gelatin solution 15% gelatin solution, 4% sodium alginate solution, 2-4 generation rBMSCs cell suspension or physiological saline are mixed uniformly in a volume ratio of 3:2:1, and the final cell concentration is 10 6 -10 7 /mL.
  • the addition amount is 1.0 mg/mL.
  • Step 3 Preparation and repair of bone defect model
  • Eight-week-old male SD rats were anesthetized to create a 2.5mm diameter and 1mm deep circular defect on the inner side of the proximal tibia.
  • the experimental group was a gel-cell mixture containing magnesium metal particles
  • the control group was a gel containing magnesium metal particles without stem cells.
  • the defect was injected with a gel mixture of 20 ⁇ L per sample. After the injection, a 0.1 mol/L CaCl 2 solution was injected for cross-linking, and physiological saline was gently rinsed 3 times. After 2 weeks, the rats were sacrificed, the materials were taken, fixed, and the new bone was analyzed by three-dimensional reconstruction of MicroCT scan; paraffin embedding, section HE staining, and statistical analysis of new bone growth.
  • the present invention prepares a hydrogel containing living cells, injectable, in-situ pores, and biological activity based on the principle of gas generation by the degradation of magnesium metal particles.
  • the in vivo and in vitro survival rate of the cells contained in the gel is measured. Test; and test the effect of the gel on promoting tissue regeneration and repair on a rat bone defect model.
  • the internal porous structure of the gel formed by the degradation of magnesium metal particles can facilitate the penetration of oxygen and nutrients, and improve the survival rate of stem cells; at the same time, the porous structure is also beneficial to induce the rapid growth of blood vessels in external tissues and accelerate the vascularization process; , Magnesium ions generated by the degradation of magnesium metal particles in the body act on stem cells to promote the proliferation and differentiation of cells in the osteogenic direction, accelerate the formation of new bone, and promote the repair of bone defects.

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Abstract

一种可注射原位生孔水凝胶体系及其制备方法和用途，所述可注射原位生孔水凝胶体系以可注射水凝胶作为连续基底相，分离的活细胞和镁金属颗粒分布于连续基底相中，所述可注射水凝胶为水凝胶的前驱体或者预聚物，通过交联后可形成水凝胶。所述可注射原位生孔水凝胶体系可在凝胶包载活细胞同时制孔，将水凝胶的可注射性和多孔结构良好结合，对于腔隙性、手术难操作、不规则缺损组织的修复有重要意义；同时镁金属颗粒产气降解后生成镁离子，可提高凝胶的生物活性，促进组织修复。

Description

一种可注射原位生孔水凝胶体系及其制备方法和用途 技术领域
本发明涉及一种水凝胶领域，特别是涉及一种生物活性水凝胶及其制备方法。
背景技术
水凝胶由于其与天然细胞外基质高度相似的仿生结构，在组织工程和再生医学领域中得到了广泛应用，可作为活细胞三维培养及运载系统。与传统组织工程支架材料相比，可注射水凝胶体系可在微创条件下将干细胞运输至缺损区，操作简便高效。然而有研究报道随水凝胶植入缺损区的干细胞只有1-20％可在宿主体内存活，这是由于水凝胶的高分子聚合网络结构中保持着大量水分，无相互贯通的多孔结构，不利于营养物质的快速扩散及周围血管长入，这极大地影响了负载其中的细胞的生物活性及生存状态，成为扩大水凝胶组织再生应用的一大障碍。
具有多孔结构的水凝胶可通过传统的模板法、冷冻干燥法、相分离法等制备，但这类方法制备的凝胶均为预成支架型，失去了凝胶的可注射性这一特性，限制了其应用范围，同时一些方法条件严苛，过程复杂，对活细胞不利，无法在载细胞同时制孔。中国专利CN103237565B公开了一种使用氧化海藻酸盐微球作为制孔剂的方法，同时包载活细胞和制孔微球的水凝胶在注射至体内后微球降解，在凝胶内留下空泡孔隙。这种“原位成孔”的方法使凝胶在注射区内直接生成孔隙结构，从而使凝胶同时具备可注射性和多孔结构。但该方法中微球的降解依赖于长时间组织液的渗透接触，在有时间限定的条件下会导致凝胶内部微球降解不均一、孔隙不连通，不利于凝胶深部细胞的存活及快速血管化，在报道中亦发现该多孔凝胶在促进骨再生方面效果并不显著。除此之外，目前尚未有其它方法可在包载活细胞的可注射水凝胶中制备多孔结构。
发明内容
鉴于以上所述现有技术的缺点，本发明的目的在于提供一种可注射原位生孔水凝胶体系及其制备方法和用途，用于解决现有技术中的问题。
为实现上述目的及其他相关目的，本发明是通过以下技术方案获得的。
本发明提供一种可注射原位生孔水凝胶体系，所述可注射原位生孔水凝胶体系以可注射水凝胶作为连续基底相，分离的活细胞和镁金属颗粒分布于连续基底相中，所述可注射水凝胶 为水凝胶的前驱体或者预聚物，通过交联后可形成水凝胶。
根据本发明技术方案，所述可注射水凝胶选自藻酸盐、明胶、琼脂糖、壳聚糖、胶原、丝素蛋白、纤维素、葡萄糖、透明质酸、硫酸软骨素、基质胶、聚乙烯醇和聚乙二醇及它们的衍生物中的一种或多种水凝胶的前驱体或预聚物。
根据本发明技术方案，分离细胞为选自间充质干细胞、胚胎干细胞、诱导性多功能细胞或成体细胞的一种或多种组合。
根据本发明技术方案，分离的活细胞可通过组织块贴壁法、机械分离法、消化分离法、悬浮培养法、流式细胞分选法、免疫磁珠分选法获得。
根据本发明技术方案，所述可注射水凝胶体系中细胞浓度为10 6-10 7个/mL。
根据本发明技术方案，所述镁金属颗粒的平均粒径为20μm～100μm。最大粒径为150μm。所述镁金属颗粒包括机械研磨法、雾化法、还原法、电解法等得到的片状颗粒、球形颗粒。所述镁金属颗粒中镁元素质量分数超过99％。优选地，所述镁金属颗粒中镁元素质量分数超过99.98％。
根据本发明技术方案，所述镁金属颗粒在可注射水凝胶体系中的添加量为0.2mg/mL～2.0mg/mL。
本发明还公开了制备如上述所述的可注射原位生孔水凝胶体系的方法，将可注射水凝胶、分离的活细胞和镁金属颗粒混合。
本发明还公开了如上述所述的可注射原位生孔水凝胶体系在制备组织再生修复剂的用途。
根据本发明技术方案，在使用时，可注射原位生孔水凝胶体系在组织再生修复处交联形成多孔水凝胶。
根据本发明技术方案，联水凝胶的方法选自化学交联、物理交联和化学物理混合交联中的一种。
根据本发明技术方案，所述交联为采用离子交联、温度控制、酸碱反应、光引发和聚合反应中的一种或多种。具体地，离子交联即使用含有钙离子、锌离子、锶离子、二价铁离子、三聚磷酸根离子、硫酸根离子、柠檬酸根离子其中一种离子的水溶液作为交联剂；温度控制即使用4～25℃去离子水水浴冷却或37℃去离子水水浴升温；酸碱反应即使用盐酸或氢氧化钠水溶液作为交联剂；光引发即使用波长大于300nm的紫外光引发交联反应，或同时借助1-[4-(2-羟乙氧基)-苯基]-2-羟基-2甲基丙酮(Irgacure 2959)或2,2-二甲氧基-2-苯基苯乙酮(Irgacure 651)作为光引发剂；聚合反应即使用乙二醛、聚丙烯酸、聚乙二醇、草酸、β-磷酸甘油、京尼平、水溶性碳二亚胺其中一种作为交联剂。
根据本发明技术方案，交联的时间为10s～30min。这一交联时间刚好与待修复组织的可操作时间一致，使得本申请中的可注射原位生孔水凝胶体系可用作这些组织部位的修复剂。
根据本发明技术方案，所述可注射原位生孔水凝胶体系交联后达到溶胀平衡时弹性模量为0.1kPa～100kPa，含水量超过80wt％。
根据本发明技术方案，所述可注射原位生孔水凝胶体系交联后形成的孔隙结构的平均直径为100μm～300μm和/或最大直径为500μm。
根据本发明技术方案，多孔水凝胶的孔隙率不少于50％。优选为50％～80％。
本发明上述技术方案解决了现有技术中在包载活细胞的可注射水凝胶中制备多孔结构的困难，提出在水凝胶中添加镁金属颗粒，利用镁金属颗粒遇水降解反应产生的氢气发泡制孔，气体在镁金属颗粒周围形成，逐渐膨大至相互接触融合，最终突破凝胶限制而逸出，在凝胶中留下相互连接且与外界相通的孔隙结构。该方法在普遍高含水的水凝胶中具有可行性及普适性，且反应温和，无需额外条件，对活细胞友好。此外，镁金属颗粒降解生成的镁离子具有促细胞增殖分化，促进成骨成血管功能，使水凝胶同时具有生物活性。
本发明上述技术方案具有以下有益效果：
1)本发明通过镁金属颗粒制孔形成的多孔可注射水凝胶体系具有多孔结构，有利于氧气、营养物质的快速渗入，从而利于周围血管组织的长入，有效提高包载于凝胶中的细胞的存活率，为解决再生修复研究中细胞体内存活率低的问题提供一种新的技术方案。
2)本发明制孔方式简单，步骤简明，未引入毒性物质，对活细胞友好。
3)本发明将水凝胶的可注射性和多孔结构良好结合，对于腔隙性、手术难操作、不规则缺损组织的修复有重要意义；同时镁金属颗粒产气降解后生成镁离子，可提高凝胶的生物活性，对干细胞有成骨向分化诱导作用，可促进新骨形成，在骨缺损修复中有应用价值。
附图说明
图1为镁金属颗粒在多种水凝胶中制孔的体式显微镜及X线片表征多孔结构效果图，并附有体式显微镜放大图以观察孔隙贯通情况。其中，A为Ⅰ型胶原，通过酸碱反应成胶；B为明胶，通过降温冷却成胶；C为丝素蛋白，通过聚合反应交联；D为海藻酸钠(茜素红染色)，通过钙离子交联。
图2为不同规格及不同添加量镁金属颗粒在明胶-海藻酸钠可注射水凝胶体系中制孔情况。其中，A为相同添加量下粒径100μm切屑镁金属颗粒及粒径20μm、50μm、100μm雾化球形镁金属颗粒制孔情况的MicroCT表征效果图；B为相同颗粒规格条件下不同镁金属颗 粒添加量(0、0.2、1.0、2.0mg/mL)制孔情况扫描电镜表征效果图。
图3为镁金属颗粒制孔获得的多孔水凝胶中包载活细胞并提高其存活率的研究结果。其中，A为明胶-海藻酸钠凝胶包载3T3-L1细胞体外培养，活死双染色检测镁金属颗粒制孔对细胞体外存活率的影响；B为明胶-海藻酸钠凝胶包载Luciferase及GFP双标记的细胞植入裸鼠皮下，活体荧光成像评估镁金属颗粒制孔对细胞体内存活率的影响。
图4为镁金属颗粒制孔的水凝胶体系利于周围血管、组织长入的实验效果图，Microfil灌注，MicroCT扫描评估血管长入数目，切片HE染色观察组织长入量。
图5为镁金属颗粒制孔的水凝胶体系利于血管化骨再生修复的实验效果图，海藻酸钠-明胶凝胶包载大鼠骨髓间充质干细胞修复大鼠股骨远端缺损，MicroCT评估成骨情况。
图6为镁金属颗粒在包载活细胞的水凝胶体系中制孔利于骨缺损修复的实验效果图，海藻酸钠-明胶凝胶中加入镁金属颗粒，同时包载或不包载大鼠骨髓间充质干细胞修复大鼠胫骨缺损，MicroCT及切片HE染色评估成骨情况。
具体实施方式
以下由特定的具体实施例说明本发明的实施方式，熟悉此技术的人士可由本说明书所揭露的内容轻易地了解本发明的其他优点及功效。
在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围。下列实施例中未注明具体条件的试验方法，通常按照常规条件，或者按照各制造商所建议的条件。
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
实施例1
本实施例为使用镁金属颗粒制孔形成的多孔可注射水凝胶体系的制备与表征
步骤一、使用镁金属颗粒作为制孔剂制备多孔可注射水凝胶
Ⅰ型鼠尾胶原制备：3mg/mL胶原300μL，10xPBS 50μL，dH 2O 81μL，0.1mol/L NaOH溶液69μL，加入镁金属颗粒混合，室温静置成胶。
实验结果如图1A所示，与对照组相比，添加镁金属颗粒可在胶原成胶同时在凝胶内形成孔隙。
15％(w/v)明胶溶液水浴升温至37℃，加入镁金属颗粒混合，降温至室温25℃成胶。
实验结果如图1B所示，与对照组相比，添加镁金属颗粒可在明胶成胶同时在凝胶内形成孔隙。
8％(w/v)丝素蛋白溶液、0.5wt％H 2O 2溶液、1kU辣根过氧化物酶按1000：20：20体积比配制，加入镁金属颗粒混合，37℃静置5-10min成胶。
实验结果如图1C所示，与对照组相比，添加镁金属颗粒可在丝素蛋白成胶同时在凝胶内形成孔隙。
以0.5％(w/v)NaCl溶液作为溶剂，配制15％(w/v)明胶溶液、4％(w/v)海藻酸钠溶液。将15％明胶溶液、4％海藻酸钠溶液、1mg/mL茜素红溶液按3:2:1体积比混合均匀，加入镁金属颗粒混合，溶液倒入圆柱形模具，用0.1mol/L CaCl 2溶液交联。凝胶脱模后用体式显微镜拍照观察，并拍摄X线片。
实验结果见图1D，镁金属颗粒可在明胶-海藻酸钠凝胶内部形成多层次孔隙结构、孔隙相互贯通。
步骤二、使用镁金属颗粒作为制孔剂的参数优化
分别使用粒径100μm研磨片状镁金属颗粒及粒径20μm、50μm、100μm雾化球形镁金属颗粒按相同添加量加入步骤一所述明胶-海藻酸钠混合溶液中，成型模具及成胶方式同步骤一。凝胶脱模后MicroCT扫描重建。
实验结果见图2A，与其它规格镁金属颗粒相比，粒径20μm雾化球形镁金属颗粒在明胶-海藻酸钠混合水凝胶中制孔的凝胶孔隙率最高。
使用粒径20μm雾化球形镁金属颗粒按0.2、1.0、2.0mg/mL含量添加入步骤一所述明胶-海藻酸钠混合液中，对照组不添加镁金属颗粒，成型模具及成胶方式同步骤一。凝胶脱模后置入液氮中快速冷冻30min，取出后于冷冻干燥机中抽真空4小时，得到干燥样品后喷金，场发射扫描电子显微镜进行样品表征，扫描50倍、100倍视野。
实验结果见图2B，随着镁金属颗粒添加量的增加，明胶-海藻酸钠混合水凝胶内部孔隙率增加，其中1mg/mL添加量形成的孔隙较均一，孔隙尺寸在100～300μm。
实施例2
本实施例为使用镁金属颗粒制孔的多孔水凝胶提高细胞的存活率。
步骤一、使用镁金属颗粒作为制孔剂制备多孔水凝胶提高细胞体外存活率
配制15％明胶溶液、4％海藻酸钠溶液。将15％明胶溶液、4％海藻酸钠溶液及3T3-L1细胞悬液按3:2:1体积比混合均匀，细胞终浓度10 6-10 7/mL，实验组加入镁金属颗粒混合，对照组无镁金属颗粒，0.1mol/L CaCl 2溶液交联，每样品150μL体积。体外培养1、3、7天，活死双染色试剂盒染色细胞，荧光显微镜观察绿色及红色细胞数量。
实验结果见图3A，体外培养结果显示在含细胞的水凝胶中使用镁金属颗粒原位制孔并未造成更多细胞死亡，相反多孔结构利于细胞的存活，与对照组有显著差异。
步骤二、使用镁金属颗粒作为制孔剂制备多孔可注射水凝胶提高细胞体内存活率
构建LV-EF1A>Luciferase-CMV>EGFP/T2A/Puro慢病毒载体，转染3T3-L1细胞。荧光显微镜观察绿色荧光蛋白表达，检测细胞转染效率。扩增Luciferase和GFP双标记的细胞。配制15％明胶溶液、4％海藻酸钠溶液。将15％明胶溶液、4％海藻酸钠溶液及上述细胞悬液按3:2:1体积比混合均匀，细胞终浓度10 6-10 7/mL，实验组加入镁金属颗粒混合，对照组无镁金属颗粒，0.1mol/L CaCl 2溶液交联，每样品150μL体积。上述含细胞凝胶样品植入裸鼠背部皮下，3、10天时按150mg/kg的量，每只裸鼠腹腔注射荧光素钾盐，15min后注射麻醉裸鼠，活体成像分析荧光强度。
实验结果见图3B，活体荧光成像显示细胞随凝胶植入体内后在镁金属颗粒制孔组生存率更高且可不断增殖，说明镁金属颗粒制孔促进细胞存活的效果在体内可维持。
实施例3
使用镁金属颗粒制孔的多孔可注射水凝胶促进血管、组织长入。
15％明胶溶液、4％海藻酸钠溶液、0.5％NaCl溶液按3:2:1体积比混合均匀，实验组加入镁金属颗粒混合，对照组无镁金属颗粒，凝胶注入八周龄雄性SD大鼠背部皮下，每样200μL，注入后用0.1mol/L CaCl 2溶液注射交联，生理盐水冲洗洗涤3次。1周后行降主动脉插管Microfil血管灌注。取材，固定，MicroCT扫描，三维重建，分析血管数目。样品扫描完成后 石蜡包埋，切片，HE染色，统计分析组织长入量。
实验结果见图4，在镁金属颗粒制孔的多孔凝胶组，周围血管、组织长入量都显著高于无孔组。
实施例4
使用镁金属颗粒制孔的多孔水凝胶包载干细胞促进血管化骨再生。
步骤一、大鼠骨髓干细胞(rBMSCs)分离培养
4周龄SD大鼠脱脱颈处死，无菌条件下分离双侧股骨、胫骨，剪掉双侧干骺端，使用DMEM培养液反复冲洗骨髓腔，收集冲洗液室温1000rpm离心15分钟后弃上清，细胞重悬，接板培养。
步骤二、含rBMSCs的凝胶制备
15％明胶溶液、4％海藻酸钠溶液、2-4代rBMSCs细胞悬液按3:2:1体积比混合均匀，细胞终浓度为10 6-10 7/mL。
步骤三、骨缺损模型制备及修复
八周龄雄性SD大鼠麻醉后股骨远端内侧制造2.5mm直径、深度3mm圆柱形缺损。实验组在凝胶-细胞混合液中加入镁金属颗粒，对照组无镁金属颗粒。缺损处注射凝胶-细胞混合液，每样30μL，注射后0.1mol/L CaCl 2溶液注射交联，生理盐水轻力冲洗3次，同时设空白对照组。3周后处死大鼠，取材，固定，MicroCT扫描三维重建分析新生骨情况。
实验结果如图5所示，使用含镁多孔凝胶修复的缺损新骨再生效果最佳，与空白对照组及无镁金属颗粒组有明显差异。
实施例5
使用镁金属颗粒在包载干细胞的水凝胶中原位生孔提高缺损修复效果。
步骤一、大鼠骨髓干细胞(rBMSCs)分离培养
4周龄SD大鼠脱脱颈处死，无菌条件下分离双侧股骨、胫骨，剪掉双侧干骺端，使用DMEM培养液反复冲洗骨髓腔，收集冲洗液室温1000rpm离心15分钟后弃上清，细胞重悬，接板培养。
步骤二、含镁金属颗粒可注射凝胶制备
15％明胶溶液、4％海藻酸钠溶液、2-4代rBMSCs细胞悬液或生理盐水按3:2:1体积比混合均匀，细胞终浓度为10 6-10 7/mL，镁金属颗粒的添加量为1.0mg/mL。
步骤三、骨缺损模型制备及修复
八周龄雄性SD大鼠麻醉后胫骨近端内侧制造2.5mm直径、深度1mm圆形缺损。实验组为含镁金属颗粒的凝胶-细胞混合液，对照组为含镁金属颗粒无干细胞的凝胶。缺损处注射凝胶混合液，每样20μL，注射后0.1mol/L CaCl 2溶液注射交联，生理盐水轻力冲洗3次。2周后处死大鼠，取材，固定，MicroCT扫描三维重建分析新生骨情况；石蜡包埋，切片HE染色，统计分析新骨生长量。
实验结果如图6所示，与无细胞的单纯多孔水凝胶相比，使用镁颗粒在包载活细胞的水凝胶中原位生孔可有效提高骨再生效果。
综上所述，本发明基于镁金属颗粒降解产气原理制备包载活细胞、可注射、原位生孔、生物活性的水凝胶，对凝胶所包载细胞的体内体外存活率进行了检测；并在大鼠骨缺损模型上检测该凝胶对于促进组织再生修复的效果。研究结果表明：镁金属颗粒降解形成的凝胶内部多孔结构可利于氧气及营养物质的渗入，提高干细胞的生存率；同时多孔结构也有利于诱导外部组织血管快速长入，加速血管化进程；此外，镁金属颗粒体内降解生成的镁离子作用于干细胞，促进细胞成骨向的增殖、分化，加速新骨的形成，促进骨缺损的修复。
上述实施例仅例示性说明本发明的原理及其功效，而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下，对上述实施例进行修饰或改变。因此，举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变，仍应由本发明的权利要求所涵盖。

Claims (16)

  1. 一种可注射原位生孔水凝胶体系，其特征在于，所述可注射原位生孔水凝胶体系以可注射水凝胶作为连续基底相，分离的活细胞和镁金属颗粒分布于连续基底相中；所述可注射水凝胶为水凝胶的前驱体或者预聚物，通过交联后可形成水凝胶。
  2. 根据权利要求1所述的可注射原位生孔水凝胶体系，其特征在于，所述可注射水凝胶选自藻酸盐、明胶、琼脂糖、壳聚糖、胶原、丝素蛋白、纤维素、葡萄糖、透明质酸、硫酸软骨素、基质胶、聚乙烯醇和聚乙二醇及它们的衍生物中的一种或多种水凝胶的前驱体或预聚物。
  3. 根据权利要求1所述的可注射原位生孔水凝胶体系，其特征在于，分离细胞为选自间充质干细胞、胚胎干细胞、诱导性多功能细胞或成体细胞的一种或多种组合。
  4. 根据权利要求1所述的可注射原位生孔水凝胶体系，其特征在于，所述可注射原位生孔水凝胶体系中分离的活细胞的浓度为(10 6-10 7)个/mL。
  5. 根据权利要求1所述的可注射原位生孔水凝胶体系，其特征在于，包括如下特征中的一种或两种：所述镁金属颗粒的平均粒径为20μm～100μm；最大粒径为150μm。
  6. 根据权利要求1所述的可注射原位生孔水凝胶体系，其特征在于，所述镁金属颗粒在可注射原位生孔水凝胶体系中的添加量为0.2mg/mL～2.0mg/mL。
  7. 根据权利要求1所述的可注射原位生孔水凝胶体系，其特征在于，所述镁金属颗粒中镁元素质量分数超过99.98％。
  8. 一种制备如权利要求1～7任一项所述的可注射原位生孔水凝胶体系的方法，将可注射水凝胶、分离的活细胞和镁金属颗粒混合。
  9. 如权利要求1～7任一项所述的可注射原位生孔水凝胶体系在制备组织再生修复剂的用途。
  10. 根据权利要求9所述的用途，其特征在于，在使用时，可注射原位生孔水凝胶体系在组织再生修复处交联形成多孔水凝胶。
  11. 根据权利要求10所述的用途，其特征在于，交联水凝胶的方法选自化学交联、物理交联和化学物理混合交联中的一种。
  12. 根据权利要求11所述的用途，其特征在于，所述交联的方法为采用离子交联、温度控制、酸碱反应、光引发和聚合反应中的一种或多种。
  13. 根据权利要求10所述的用途，其特征在于，交联的时间为10s～30min。
  14. 根据权利要求10所述的用途，其特征在于，所述可注射原位生孔水凝胶体系交联后达到溶胀平衡时弹性模量为0.1kPa～100kPa，含水量超过80wt％。
  15. 根据权利要求10所述的用途，其特征在于，所述可注射原位生孔水凝胶体系交联后 形成的孔隙结构的平均直径为100μm～300μm和/或最大直径为500μm。
  16. 根据权利要求10所述的用途，其特征在于，多孔水凝胶的孔隙率不少于50％。
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