CN110760103A - Viscoelastic hydrogel and preparation method and application thereof - Google Patents
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Abstract
The viscoelastic hydrogel disclosed by the invention has good viscoelasticity and high mechanical strength, can well protect living cells in the gel, can be used as a cell loading medium, also has good shear thinning property and good injectability, and can be used for preparing cell injection medium materials, cell-loaded 3D printing materials, drug carrier materials, cell protection materials and the like; meanwhile, the preparation method is simple, the raw materials are cheap and easy to obtain, the actual operation is convenient and rapid, the storage condition is not severe, and the method is suitable for popularization and application and mass production.
Description
Technical Field
The invention relates to the field of injectable hydrogel, in particular to viscoelastic hydrogel and a preparation method and application thereof.
Background
Articular cartilage defect is a common disease in clinic, and cartilage tissue is in an environment without blood vessels, nerves and lymphatic return, has limited self-repair capability, and is difficult to regenerate once being damaged.
The traditional treatment methods comprise autologous cell transplantation, microfracture, joint replacement and the like, wherein the application of the autologous cell transplantation method is greatly limited due to limited cell sources and easy damage or dysfunction of the material-taking parts; the cartilage formed by the micro-fracture scheme is fibrous, the normal cartilage tissue is composed of hyaline cartilage, the fibrous cartilage has poor mechanical property and cannot bear the mechanical load of joints, so that the repair capability is reduced; the traditional joint replacement surgery may cause secondary injury, has a large renovation risk and has unsatisfactory treatment effect.
Fortunately, tissue engineering provides a new direction for repairing cartilage defects, wherein scaffolds in the tissue engineering comprise three elements, namely cells, scaffolds and regulatory factors, researchers have proved that the scaffolds have great potential in repairing and replacing damaged or necrotic tissues and organs of human bodies, however, the implantation of the scaffolds usually requires open surgery, which results in large wound area and may cause serious infection and clinical surgical complications clinically, thereby reducing the repairing effect.
With the development of clinical minimally invasive arthroscopic surgery and cell therapy, many researchers have proposed tissue repair by injecting cells into the defect site with a syringe, the cells being injected as a cell suspension in a buffer. However, during the injection process, the cells are subjected to different levels of shear stress, pressure, etc. at different locations of the injector, which can lead to massive cell death. In addition, a small number of cells that can be precisely localized to the defect are also in a free state and are likely to be washed away by the synovial fluid, thereby reducing the therapeutic effect.
Therefore, cell therapy has limited use in minimally invasive cartilage regeneration due to the lack of a suitable injection medium.
Disclosure of Invention
The invention mainly solves the technical problem of providing a viscoelastic hydrogel, a preparation method and application thereof, wherein the viscoelastic hydrogel has viscoelasticity and can protect cells.
In order to solve the technical problems, the invention adopts a technical scheme that:
providing viscoelastic hydrogel, which comprises 4-6 mg/mL aldehyde group modified cellulose nanocrystal, 4-6 mg/mL collagen and water; the aldehyde group modified cellulose nanocrystal is a substance obtained after at least one hydroxyl group in the cellulose nanocrystal structure is oxidized into aldehyde group.
The inventor unexpectedly finds that the hydrogel prepared by mixing the aldehyde-group modified cellulose nanocrystal and the collagen has good shear thinning capability and good injectability and self-healing performance, and the aldehyde group in the aldehyde-group modified cellulose nanocrystal and the amino group in the collagen can react with Schiff base (imine) bonds, so that the Schiff base and aldehyde and amine reactants are in dynamic balance.
Because the reaction in the hydrogel is in dynamic balance, and the real-time concentration of the raw material and the product is difficult to measure, in the invention, the concentration of the aldehyde-group modified cellulose nanocrystal or the collagen in the viscoelastic hydrogel refers to the concentration obtained by dividing the mass of the aldehyde-group modified cellulose nanocrystal or the collagen in the raw material by the total volume of the hydrogel.
Surprisingly, the inventor also finds that the hydrogel prepared by combining the aldehyde-group modified cellulose nanocrystal and the collagen has good viscoelasticity, particularly higher elastic modulus and stronger stress relaxation capacity, and can resist stress to protect substances in the gel under the condition that the gel is subjected to external stress.
Further, the aldehyde-modified cellulose nanocrystal is a substance obtained by oxidizing 2, 3-position hydroxyl groups in part or all of glucose units of the cellulose nanocrystal into aldehyde groups.
In the invention, NaIO is adopted42, 3-hydroxy in the cellulose nanocrystal is oxidized into aldehyde group, and the reaction principle is as follows:
further, the aldehyde group content of the aldehyde group modified cellulose nanocrystal is 10-20%.
The aldehyde group content refers to the proportion of oxidized glucose units in the glucose units of the aldehyde group-modified cellulose nanocrystal.
The collagen is type I collagen, preferably bovine type I collagen.
In a specific embodiment of the present invention, the aldehyde-modified cellulose nanocrystal is: the mass ratio of the collagen is 0.8-1.2.
The invention also provides a preparation method of the viscoelastic hydrogel, which comprises the following steps: mixing the aldehyde group modified cellulose nanocrystalline solution with the collagen solution to obtain the collagen nano-composite material.
In a specific embodiment of the present invention, the aldehyde-modified cellulose nanocrystal solution or the collagen solution is prepared from PBS buffer.
Further, the concentration of the aldehyde group modified cellulose nanocrystalline solution is 8-12 mg/mL, and the concentration of the collagen solution is 8-12 mg/mL.
Further, the pH value of the aldehyde group modified cellulose nanocrystalline solution is 5-8, and the pH value of the collagen protein solution is 5-8.
In the specific embodiment of the invention, aldehyde modified cellulose nanocrystalline solution and collagen solution are mixed to prepare the viscoelastic hydrogel, two syringe injectors are adopted to respectively absorb the aldehyde modified cellulose nanocrystalline solution and the collagen solution, and the injectors are pushed to inject into the same area to mix the aldehyde modified cellulose nanocrystalline solution and the collagen solution.
In the preparation of the viscoelastic hydrogel, the aldehyde-based modified cellulose nanocrystalline solution and the collagen solution are not limited to be mixed by the syringe injector, and all devices with the same function or capable of achieving the same purpose can be suitable for the viscoelastic hydrogel, such as a double-syringe injector and the like.
The invention also provides application of the viscoelastic hydrogel in preparation of one or more of injection medium materials, drug carrier materials and cell protection materials.
The viscoelastic material has good viscoelasticity, high mechanical strength and good injectability, and can be used as a medium material for transporting or transferring loaded substances such as injection medium materials or drug carrier materials.
The substance loaded in the injection medium material or the drug carrier material can be solid or liquid effective substances such as chemical drugs, various extracts, cells, fillers (such as bone repair materials) and the like which are added according to the use or efficacy requirements.
Experiments prove that the viscoelastic hydrogel disclosed by the invention has good viscoelastic property and high and low mechanical strength, can better protect cells and improve the survival rate of the cells in a medium, and can be used for preparing materials needing to protect the survival of the cells and used in the processes of cell transfer, transfer and the like.
Further, the injection medium is a cell injection medium material.
The invention has the beneficial effects that:
(1) the aldehyde group modified cellulose nanocrystalline and collagen compounded hydrogel has good viscoelasticity, high mechanical strength and injectability, can protect substances in the gel, and can be used for preparing injection medium materials, drug carrier materials, cell protection materials and the like.
(2) The hydrogel disclosed by the invention has good viscoelasticity and high mechanical strength, can well protect living cells in the gel, can be used as a cell loading medium, and can be used for preparing a cell injection medium material, a cell loaded 3D printing material and the like.
(3) The hydrogel preparation method disclosed by the invention is simple, the raw materials are cheap and easy to obtain, expensive instruments and equipment are not needed, the danger coefficient of the preparation method is low, the actual operation is convenient and quick, a large amount of injectable hydrogel with good viscoelasticity can be prepared in a short time, the storage condition is not severe, and the hydrogel preparation method is suitable for popularization and application and mass production.
Drawings
FIG. 1 is an infrared spectrum of CNC, a-CNC, collagen and a-CNC/collagen hydrogels;
figure 2 is an SEM image of two hydrogel cross-sections: (A) CNC/collagen hydrogel, (B) a-CNC/collagen hydrogel;
figure 3 is the compression properties of two hydrogels: (A) stress-strain curves for the two hydrogels, (B) the elastic moduli of the two hydrogels;
figure 4 is the rheological properties of two hydrogels: (A) storage moduli of the two hydrogels, (B) viscosity flow curves of the two hydrogels, (C) stress relaxation curves of the two hydrogels, and (D) stress relaxation times of the two hydrogels;
figure 5 is the injection performance of two hydrogels: (A) CNC/collagen hydrogel, (B) a-CNC/collagen hydrogel;
FIG. 6 is confocal imaging after FDA/PI staining following injection of two hydrogel-loaded hydrogels: (A) a-CNC/collagen hydrogel, (B) CNC/collagen hydrogel.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
1. Preparing aldehyde group modified cellulose nanocrystalline solution:
1) weighing equal mass of Cellulose Nanocrystals (CNC) and NaIO4。
2) Enough deionized water is taken to dissolve CNC and NaIO4Dilute sulfuric acid (concentration 0.1mol/L) was added to adjust pH 3.
3) And (3) carrying out water bath at the constant temperature of 40 ℃ in a dark condition, stirring simultaneously, and reacting for 3 h.
4) After the reaction, the sample was centrifuged with deionized water and dialyzed with a 4000 mesh dialysis bag for three days (deionized water was changed every 2 h) until no IO was detected3 —And (4) residual: firstly, dripping a small amount of centrifuged supernatant on starch potassium iodide test paper, and dripping a small amount of dilute sulfuric acid until the test paper does not change color; the dialyzed sample was titrated to determine the aldehyde content of 16.27%.
5) And (3) freeze-drying the sample to obtain the aldehyde group modified cellulose nanocrystalline solid (a-CNC).
6) Dissolving the aldehyde modified cellulose nanocrystalline solid (a-CNC) into PBS with a certain volume to prepare an a-CNC solution with the a-CNC concentration of 10mg/mL, and then adjusting the pH value of the a-CNC solution to 7.
2. Preparation of collagen solution:
dissolving collagen into PBS with a certain volume to obtain collagen solution with collagen concentration of 10mg/mL, adjusting pH of the mixed solution to 7, and standing for half an hour in ice bath for prepolymerization to obtain collagen solution.
3. Preparation of viscoelastic injectable hydrogel:
and (3) respectively taking the collagen solution and the a-CNC solution with the same volume by using two syringe injectors at room temperature, pushing the injectors to mix the two solutions in a container, slowly mixing for half a minute to fully mix the two solutions uniformly, and reacting to obtain the a-CNC/collagen hydrogel.
Example 2
1. Preparing aldehyde group modified cellulose nanocrystalline solution:
1) weighing equal mass of Cellulose Nanocrystals (CNC) and NaIO4。
2) Enough deionized water is taken to dissolve CNC and NaIO4Dilute sulfuric acid (concentration 0.1mol/L) was added to adjust pH 3.
3) And (3) carrying out water bath at the constant temperature of 40 ℃ in a dark condition, stirring simultaneously, and reacting for 3 h.
4) After the reaction, the sample was centrifuged with deionized water and dialyzed with a 4000 mesh dialysis bag for three days (deionized water was changed every 2 h) until no IO was detected3 —And (4) residual: firstly, a small amount of potassium iodide is dripped on starch potassium iodide test paperDripping a small amount of dilute sulfuric acid into the supernatant liquor after the heart till the test paper does not change color; the dialyzed sample was titrated to determine the aldehyde content of 16.27%.
5) And (3) freeze-drying the sample to obtain the aldehyde group modified cellulose nanocrystalline solid (a-CNC).
6) Dissolving the aldehyde modified cellulose nanocrystalline solid (a-CNC) into PBS with a certain volume to prepare an a-CNC solution with the a-CNC concentration of 8mg/mL, and then adjusting the pH value of the a-CNC solution to 5.
2. Preparation of collagen solution:
dissolving collagen into PBS with a certain volume to obtain collagen solution with collagen concentration of 8mg/mL, adjusting pH of the mixed solution to 8, and standing for half an hour in ice bath for prepolymerization to obtain collagen solution.
3. Preparation of viscoelastic injectable hydrogel:
and (3) respectively taking the collagen solution and the a-CNC solution with the same volume by using two syringe injectors at room temperature, pushing the injectors to mix the two solutions in a container, slowly mixing for half a minute to fully mix the two solutions uniformly, and reacting to obtain the a-CNC/collagen hydrogel.
Example 3
1. Preparing aldehyde group modified cellulose nanocrystalline solution:
1) weighing equal mass of Cellulose Nanocrystals (CNC) and NaIO4。
2) Enough deionized water is taken to dissolve CNC and NaIO4Dilute sulfuric acid (concentration 0.1mol/L) was added to adjust pH 3.
3) And (3) carrying out water bath at the constant temperature of 40 ℃ in a dark condition, stirring simultaneously, and reacting for 3 h.
4) After the reaction, the sample was centrifuged with deionized water and dialyzed with a 4000 mesh dialysis bag for three days (deionized water was changed every 2 h) until no IO was detected3 —And (4) residual: firstly, dripping a small amount of centrifuged supernatant on starch potassium iodide test paper, and dripping a small amount of dilute sulfuric acid until the test paper does not change color; the dialyzed sample was titrated to determine the aldehyde content of 16.27%.
5) And (3) freeze-drying the sample to obtain the aldehyde group modified cellulose nanocrystalline solid (a-CNC).
6) Dissolving the aldehyde modified cellulose nanocrystalline solid (a-CNC) into PBS with a certain volume to prepare an a-CNC solution with the a-CNC concentration of 12mg/mL, and then adjusting the pH value of the a-CNC solution to 8.
2. Preparation of collagen solution:
dissolving collagen into PBS with a certain volume to obtain collagen solution with collagen concentration of 12mg/mL, adjusting pH of the mixed solution to 5, and standing for half an hour in ice bath for prepolymerization to obtain collagen solution.
3. Preparation of viscoelastic injectable hydrogel:
and (3) respectively taking the collagen solution and the a-CNC solution with the same volume by using two syringe injectors at room temperature, pushing the injectors to mix the two solutions in a container, slowly mixing for half a minute to fully mix the two solutions uniformly, and reacting to obtain the a-CNC/collagen hydrogel.
Example 4
The preparation method is the same as example 1, except that: the concentration of the a-CNC solution is 8mg/mL, and the concentration of the collagen solution is 10 mg/mL.
Example 5
The preparation method is the same as example 1, except that: the concentration of the a-CNC solution is 12mg/mL, and the concentration of the collagen solution is 10 mg/mL.
Test example 1
The viscoelastic injectable hydrogel prepared in example 1 is subjected to mechanical property and injection property detection and cell experiment detection; and dissolving CNC (computerized numerical control), namely non-aldehyde modified cellulose nanocrystalline, into PBS (phosphate buffer solution) with a certain volume to prepare a CNC solution with the CNC concentration of 10mg/mL, adjusting the pH of the mixed solution to 7, and compounding the mixed solution with the same collagen solution by using the method in the embodiment 1 to prepare the CNC/collagen hydrogel as a control group for detection.
1. Infrared detection
Example 1 a-CNC/collagen hydrogel was prepared by mixing a same volume of collagen with an a-CNC solution at 37 ℃ and neutral pH for 30 seconds, and the a-CNC, CNC and a-CNC/collagen hydrogel prepared according to the present invention were examined by infrared spectroscopy, and the results are shown in fig. 1.
As shown in FIG. 1, a-CNC at 1720cm compared to CNC-1A new absorption peak appears on the left and right, which is the characteristic peak of aldehyde group. 1646cm appears in the collagen hydrogel-1The characteristic absorption peak at (a), which is the stretching vibration peak of the carboxylic acid carbonyl group. The alpha-CNC/collagen spectrum was 1652cm compared to pure collagen-1There is a new peak around, and the stretching vibration peak of carboxylic acid carbonyl group in collagen disappears, which confirms that collagen is successfully cross-linked with a-CNC to form Schiff base bond (imine bond), indicating that CNC has been successfully modified to a-CNC, and a-CNC/collagen hydrogel is formed.
2. Scanning electron microscope
And (3) respectively detecting the a-CNC/collagen hydrogel and the CNC/collagen hydrogel by using a scanning electron microscope, wherein the result is shown in figure 2, SEM images show the cross sections of the two hydrogels, and after freeze-drying, the a-CNC/collagen hydrogel and the CNC/collagen hydrogel both obtain a porous structure. Both gels had distributed pores, demonstrating the cross-linking phenomenon of both gels. In addition, compared to a-CNC/collagen hydrogel, CNC/collagen hydrogels are larger in pores, looser in structure, and lower in crosslink density. Cross-sectional images of a-CNC/collagen hydrogel and CNC/collagen hydrogel show interconnected porous scaffolds to facilitate moisture absorption.
3. Compression test
The composite hydrogel was formed into a cylindrical shape having a height of 3.00mm and a diameter of 10.00mm, and the prepared sample (n-3) was immersed in PBS for 48 hours to maintain the hydrogel in an equilibrium swollen state to accommodate a mechanical compression test. The elastic modulus of the hydrogels was characterized using a dynamic mechanical analyzer (DMA, TA-Q800, USA) in compression mode at room temperature. The test parameters were set to an amplitude of 40mm, a pretension of 0.002N, a fixed frequency of 1Hz, and a force trace of 105%.
The results of the test are shown in fig. 3, and it is seen from the stress-strain curve in fig. 3 that the strain of both hydrogels is proportional to the compressive stress. After fitting the linear part of the stress-strain curve, the slope at 5% strain on the curve was taken as the modulus of elasticity. The elastic modulus of the calculated a-CNC/collagen hydrogel is larger than that of the CNC/collagen hydrogel, about 0.5kPa and 0.4kPa respectively, and the elastic modulus data of the two hydrogels have significant difference.
4. Rheology experiments
After both hydrogels were prepared and allowed to equilibrate at 37 ℃ for 10 minutes, the two hydrogels were first subjected to strain and sweep experiments using rheometer MCR302 to characterize the stress relaxation and shear thinning properties of the CNC/collagen hydrogel and the a-CNC/collagen hydrogel at 37 ℃.
The frequency sweep experiment was performed at a frequency of 1 to 100Hz and a constant strain of 1%. Strain scans were performed at 1Hz with strain ranging from 0.01% to 100%. The results show that at 1% strain and 1Hz frequency, the hydrogels exhibit linear viscoelastic responses, and these conditions are used for rheological characterization of the hydrogels.
Selecting a template for stress relaxation, setting a constant value of shear strain to be 1%, and carrying out a stress relaxation experiment at 37 ℃ to respectively determine stress relaxation curves and stress relaxation times of the two hydrogels.
Selecting template 'viscosity and flow curve', setting temperature at 37 deg.C and shear rate at 0.01-100s-1The shear-thinning curves of the two were determined separately.
The test results are shown in FIG. 4, and in the frequency sweep mode, we take the average value of the storage modulus at the frequency of 1Hz, 5Hz, and 10Hz as the storage modulus of the hydrogel at the frequency. As can be seen from fig. 4(a), the storage modulus of the a-CNC/collagen hydrogel is significantly greater than that of the CNC/collagen hydrogel; fig. 4(B) shows that the a-CNC/collagen hydrogel has better shear thinning ability than the CNC/collagen hydrogel, which indicates that the a-CNC/collagen hydrogel has better injectability.
The time point corresponding to the half of the original decrease in stress on the stress relaxation curve is taken as the stress relaxation time, and the result is shown in fig. 4(C), where the a-CNC/collagen hydrogel has a shorter stress relaxation time, which indicates that the a-CNC/collagen hydrogel has a stronger stress relaxation capacity, and indicates that the a-CNC/collagen hydrogel has good viscoelasticity and can better protect cells.
5. Injection performance
The two hydrogels were placed into syringes for injection experiments, as shown in fig. 5, CNC/collagen hydrogel immediately formed many continuous micro-drop hydrogels, and were uniformly dispersed in PBS solution at 37 ℃, the platform blue dye in the hydrogel did not escape, and the PBS solution was clear. In contrast to the control, the a-CNC/collagen hydrogel stably extruded into PBS solution had no continuous breaks and no internal trypan blue dye exuded, indicating that the a-CNC/collagen hydrogel had good payload retention and better injectability. Furthermore, as shown in fig. 5, the two hydrogels exhibited different stabilities: the CNC/collagen hydrogel in the glass bottle after strong vibration and inversion is divided into a plurality of fragments, dispersed and suspended in the PBS solution, and the PBS solution of the a-CNC/collagen hydrogel has few fragments and better stability, which indicates that the a-CNC/collagen hydrogel has good injectability and stability and can better protect the contents.
6. Immediate detection of live and dead after loaded cell injection
The rabbit-derived mesenchymal stem cells are adopted for immediate live-dead detection after cell loading injection, a-CNC suspension and CNC suspension with the same concentration are respectively mixed with collagen solution and cell suspension to construct two cell-carried hydrogel solutions, the cell density of the two solutions is 500000 cells/mL, then the hydrogel is sucked by a syringe with a needle (the size of the needle is 26gauge), and then the hydrogel is injected into a silica gel mold to form a disc with the diameter of 1cm and the height of 3 mm. Immediately after the cell-filled hydrogel was injected and formed, MSCs attached to the complex hydrogel were stained with FDA/PI to identify live or dead cells. To each hydrogel disc, 2mL of assay solution was added and incubated at room temperature for 2 minutes, and then cells were imaged by Confocal Laser Scanning Microscopy (CLSM) to check the survival status of MSCs.
The experimental results are shown in fig. 6, the mesenchymal stem cells showed good activity in both the a-CNC/collagen hydrogel and the CNC/collagen hydrogel, indicating that the hydrogel is not cytotoxic. In addition, as shown in fig. 6, the amount of dead cells in the CNC/collagen hydrogel is greater than that of the a-CNC/collagen hydrogel, which indicates that the a-CNC/collagen hydrogel can better protect cells during the injection process and improve the survival rate of the cells.
In conclusion, the a-CNC/collagen hydrogel has the advantages of large elastic modulus, stronger stress relaxation capacity, excellent viscoelasticity and high mechanical strength, can better protect loaded cells, enables the cells to show higher survival rate after injection, enables rod-shaped a-CNC to be directionally arranged in the extrusion process, further strengthens a polymer network, improves mechanical stability, and has good injectability, so that the a-CNC/collagen hydrogel can be used for preparing materials such as cell injection hydrogel, cell loading medium and the like.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. The viscoelastic hydrogel is characterized by comprising 4-6 mg/mL aldehyde-group modified cellulose nanocrystals, 4-6 mg/mL collagen and water; the aldehyde group modified cellulose nanocrystal is a substance obtained after at least one hydroxyl group in the cellulose nanocrystal structure is oxidized into aldehyde group.
2. The viscoelastic hydrogel according to claim 1, wherein the aldehyde-modified cellulose nanocrystals are obtained by oxidizing 2, 3-hydroxy groups in part or all of the glucose units of the cellulose nanocrystals to aldehyde groups; further, the aldehyde group content of the aldehyde group modified cellulose nanocrystal is 10-20%;
the collagen is type I collagen, preferably bovine type I collagen.
3. The viscoelastic hydrogel according to claim 1 or 2, wherein the aldehyde-modified cellulose nanocrystals: the mass ratio of the collagen is 0.8-1.2.
4. A process for the preparation of a viscoelastic hydrogel as claimed in any one of claims 1 to 3, characterized in that it comprises the following steps: mixing the aldehyde group modified cellulose nanocrystalline solution with the collagen solution to obtain the collagen nano-composite material.
5. The preparation method according to claim 4, wherein the solvent of the aldehyde-modified cellulose nanocrystal solution or the collagen solution is PBS buffer.
6. The preparation method according to claim 4, wherein the concentration of the aldehyde-modified cellulose nanocrystal solution is 8-12 mg/mL, and the concentration of the collagen solution is 8-12 mg/mL.
7. The preparation method according to claim 4, wherein the pH value of the aldehyde-modified cellulose nanocrystal solution is 5 to 8, and the pH value of the collagen solution is 5 to 8.
8. The preparation method according to any one of claims 4 to 7, wherein two syringe injectors are used for respectively sucking the aldehyde-based modified cellulose nanocrystal solution and the collagen solution, and the injectors are pushed to inject into the same area for mixing.
9. Use of the viscoelastic hydrogel of any one of claims 1 to 3 for the preparation of one or more of an injectable medium material, a drug carrier material, a cytoprotective material.
10. The use of claim 9, wherein the injection medium is a cell injection medium material.
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