CN112225937A - Temperature-sensitive macroporous biological hydrogel and preparation method and application thereof - Google Patents
Temperature-sensitive macroporous biological hydrogel and preparation method and application thereofDownload PDFInfo
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Abstract
Description
Technical Field
The invention relates to the technical field of biomedical materials, in particular to a temperature-sensitive macroporous biological hydrogel and a preparation method and application thereof.
Background
A hydrogel is a high molecular material composed of hydrophilic polymer chains and formed by physical or chemical crosslinking. The polymer forming the three-dimensional network structure in the hydrogel acts as a skeleton, and a large amount of water is locked in the hydrogel through the action of surface tension. The network structure formed by the hydrogel can maintain the hydrophilicity of polymer molecular chains, and the gel network can keep the form insoluble in water due to the crosslinking effect (which is the current chemical industry, 2020, 49(1), 249-252). With the progress of research, the hydrogel is found to have adjustable physical and chemical properties, can be functionalized, has the characteristics of a structure similar to extracellular matrix and the like, so that the application field of the hydrogel is expanded, and particularly in the biomedical field, the macroporous hydrogel with a larger pore diameter (up to 200 mu m) has unique advantages in the biomedical tissue engineering field.
Temperature-sensitive hydrogels can undergo a phase transition from a solution phase to a gel phase when affected by a change in external temperature, and the temperature at which this phase change occurs is called the critical phase transition temperature (or gelation temperature) (sweet and sweet, et al, Shandong chemical, 2016, 45(18), 52-54). The temperature-sensitive hydrogel is taken as an intelligent hydrogel with better application prospect in the field of biomedicine, and is hopefully applied to controlled release of drugs, separation and extraction, enzyme immobilization, immunoassay and the like.
The temperature-sensitive macroporous hydrogel combines the characteristics of the temperature-sensitive hydrogel and the macroporous hydrogel, and the larger pores can provide possibility for cell loading and provide culture space for cells; the temperature sensitive property can enable the hydrogel matrix in the solution state to enter the body through an injection method, and the hydrogel is formed in the body temperature state. Due to the characteristics, the body temperature sensitive macroporous hydrogel plays an important role in the field of biological medicine.
The existing methods for preparing the macroporous hydrogel mainly comprise a foaming method, a pore-forming method and the like (Cong Wei and the like, material guide A, 2013, 27(1), 90-93, poplar, orange and the like, functional materials, 2011, 42(2), 343-346), however, the methods are difficult to be applied in the field of biological medicine due to more defects, for example, the bubble method reduces the biological safety of the macroporous hydrogel due to the fact that the residual surfactant is difficult to remove, for example, Chinese patent CN104645946A (published Japanese 2015.5.27) discloses a method for preparing the temperature-sensitive hydrogel by using a macroporous poly N-isopropylacrylamide-chitosan semi-interpenetrating network, and the method of emulsion polymerization is utilized, span-80 is used as the surfactant, N, N' -Methylene Bisacrylamide (MBA) is used as the cross-linking agent, and Ammonium Persulfate (APS) is used as the initiator, wherein the residue of the surfactant can bring about the safety problem, therefore, how to prepare the temperature-sensitive macroporous hydrogel with higher biosafety is still to be explored.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defect and the defect of lower biological safety of the existing temperature-sensitive macroporous hydrogel and providing a preparation method of the temperature-sensitive macroporous biological hydrogel, and the prepared temperature-sensitive macroporous biological hydrogel has high biological safety and the aperture can reach 200 mu m.
The invention also aims to provide a temperature-sensitive macroporous biological hydrogel.
The invention also aims to provide application of the temperature-sensitive macroporous biological hydrogel.
The above purpose of the invention is realized by the following technical scheme:
a preparation method of temperature-sensitive macroporous biological hydrogel comprises the steps of dissolving methylcellulose and collagen in a solvent to obtain a methylcellulose-collagen mixed solution, introducing air at the speed of 0.1-10L/min, and preparing the temperature-sensitive macroporous biological hydrogel at the temperature of 20-40 ℃, wherein the mass percentage concentration of the methylcellulose in the mixed solution is 8-15%, and the mass percentage concentration of the collagen in the mixed solution is 0.2-0.7%.
Methyl Cellulose (MC) is used as a water-soluble methyl cellulose biological macromolecule, is safe and nontoxic, and has temperature sensitivity; collagen is an extracellular protein, is a main component of connective tissue, comes from the inside of an organism, can be naturally degraded and is non-toxic to a human body; the temperature-sensitive hydrogel can be prepared by blending collagen and methylcellulose, and when the temperature is lower than the gelation temperature, water molecules form a solvent cage (hydration layer) around methyl groups of the methylcellulose through hydrogen bond action to prevent the aggregation of methylcellulose molecules; when the temperature rises to reach or exceed the gelation temperature, the hydrogen bonding is weakened, the cage structure is destroyed, and methyl groups on the methyl cellulose molecular chains are exposed to hydrophobic combination, so that a network structure is formed among the methyl cellulose molecules to form gel. The invention also leads gas into the mixed solution of methyl cellulose and collagen, and the gas can be remained in the solution due to the influence of viscosity and then is placed at proper temperature to form the macroporous hydrogel. The invention follows the principle of biological safety, and uses air to prepare pores without using chemical pore-forming agent and surface active agent and without the problem of residual chemical pore-forming agent and surface active agent. Therefore, the temperature-sensitive macroporous biological hydrogel disclosed by the invention not only has a large-aperture structure (up to 200 mu m), is good in temperature sensitivity, but also is safe, non-toxic and good in biological safety, and can be widely applied to the field of biological medicines.
Preferably, the speed of the introduced air is 1.2-1.5L/min.
Preferably, the method for introducing air is to pump air by using an air pump device.
Preferably, the air pump device is an inflator.
Preferably, the air pump device further includes an air pump stone.
Preferably, the diameter of the air pump stone is 2-5 cm.
Preferably, the molecular weight of the methyl cellulose is 10000-180000, and the methyl substitution degree is 2.6-5.0.
Preferably, the collagen is one or more of type I collagen-bovine achilles tendon, type II collagen-bovine cartilage, type II collagen-bovine nasal membrane and type II collagen-chicken.
Preferably, dissolving methyl cellulose in a solvent, heating and dissolving at 30-60 ℃, and cooling to room temperature to obtain a methyl cellulose solution; and adding the collagen into the methylcellulose solution, uniformly mixing at 5-40 ℃, refrigerating for 1-25 hours at 2-10 ℃ after 0.5-10 hours, and obtaining the methylcellulose-collagen mixed solution. In the process of dissolving the methyl cellulose, the water temperature needs to be adjusted to 30-60 ℃ first, which is beneficial to the dissolution of the methyl cellulose. And because the using amount of the Methylcellulose (MC) is far larger than that of the collagen, the collagen can be better dissolved and blended when being added into the methylcellulose solution.
Preferably, the methylcellulose-collagen mixed solution is freeze-dried to obtain a methylcellulose-collagen solid; dissolving the methylcellulose-collagen solid, and introducing air. The reason why the MC solution has a low concentration before freeze-drying can enable the MC and the collagen to be better blended but not to be beneficial to gelling is that the material after freeze-drying is redissolved and can be more easily gelled.
Preferably, the step of dissolving the methylcellulose-collagen solid comprises the steps of adding a solvent, dispersing at 15-50 ℃, dissolving in an ice water bath for 4-8 hours, and refrigerating at 4-6 ℃ for 6-24 hours to obtain a solution with the mass concentration of 5-10%.
Preferably, after air is introduced for 1-10 minutes, the solution is placed in a water bath at the temperature of 20-40 ℃ to form the methylcellulose-collagen temperature-sensitive macroporous hydrogel.
Preferably, the solvent is one of deionized water, ultrapure water and physiological saline.
The invention also protects the application of the temperature-sensitive macroporous biological hydrogel in preparation of tissue administration preparations, stem cell treatments and injection treatment preparations.
Compared with the prior art, the invention has the beneficial effects that:
the temperature-sensitive macroporous biological hydrogel is prepared by utilizing the methyl cellulose with good biological safety and temperature sensitivity and the collagen, and air is introduced to form a macroporous structure.
Drawings
FIG. 1 is a process flow chart of the preparation of temperature-sensitive macroporous biological hydrogel in example 1 of the present invention.
FIG. 2 is a photograph of the temperature-sensitive macroporous biohydrogel prepared in example 1.
FIG. 3 is a scanning electron micrograph of the temperature sensitive macroporous biological hydrogel prepared in example 1 after freeze drying.
FIG. 4 is a dynamic stress-strain scan curve (37 ℃) of the temperature-sensitive macroporous biohydrogel prepared in example 1.
Detailed Description
The present invention will be further described with reference to specific embodiments, but the present invention is not limited to the examples in any way. The starting reagents employed in the examples of the present invention are, unless otherwise specified, those that are conventionally purchased.
FIG. 1 is a preparation process of example 1 of the present invention: dissolving and blending methyl cellulose and collagen in a certain mass ratio, freeze-drying to obtain a methyl cellulose-collagen solid, dissolving the solid according to a certain concentration to obtain a methyl cellulose-collagen solution, blowing air into the solution by using an air pump stone connected with an air pump, and placing at a certain temperature to form the temperature-sensitive macroporous biological hydrogel.
Example 1
A preparation method of temperature-sensitive macroporous biological hydrogel comprises the following steps:
s1, weighing 1.5 g of methylcellulose (with the molecular weight of 10000 and the degree of substitution of 2.6), placing the methylcellulose in a beaker, adding 100 ml of deionized water, stirring at 60 ℃ (the rotation speed is 900 revolutions per minute) until the methylcellulose is completely dissolved, and cooling to room temperature to obtain a methylcellulose solution;
s2, weighing 0.1 g of type I collagen-bovine achilles tendon (micromolecule), adding the I collagen-bovine achilles tendon (micromolecule) into the methylcellulose solution obtained in the step S1, stirring at 40 ℃ (900 revolutions per minute) for dissolving for 10 hours, and refrigerating and stabilizing at 2 ℃ for 25 hours to obtain a methylcellulose-collagen blending system I;
s3, freeze-drying the blending system I obtained in the step S2 to obtain a methylcellulose-collagen blending material II;
s4, weighing 1.5 g of the blending material II obtained in the step S3, placing the blending material II in a beaker, adding 15 ml of deionized water, stirring at 50 ℃ (the rotating speed is 900 revolutions per minute) for dispersion, placing the mixture in an ice water bath, stirring (900 revolutions per minute) for dissolution for 8 hours, and then refrigerating and stabilizing the mixture at 4 ℃ for 24 hours to obtain a methylcellulose-collagen solution;
s5, placing an air pump stone (with the diameter of 2cm) connected with an air pump device in the solution obtained in the step S4, pumping air at the air pumping speed of 10L/min, and after 1 minute, placing the solution in a water bath at 40 ℃ to prepare the methyl cellulose-collagen temperature-sensitive macroporous biological hydrogel.
Example 2
A preparation method of temperature-sensitive macroporous biological hydrogel comprises the following steps:
s1, weighing 0.5 g of methylcellulose (with the molecular weight of 180000 and the substitution degree of 5.0), placing the methylcellulose in a beaker, adding 10 ml of deionized water, stirring at 30 ℃ (the rotating speed is 100 revolutions per minute) until the methylcellulose is completely dissolved, and cooling to room temperature to obtain a methylcellulose solution;
s2, weighing 0.01 g of type I collagen-bovine achilles tendon (macromolecule), adding the I collagen-bovine achilles tendon (macromolecule) into the methylcellulose solution obtained in the step S1, stirring at 40 ℃ (100 r/min) to dissolve for 0.5 hour, and refrigerating at 2 ℃ for 1 hour to obtain a methylcellulose-collagen blending system I;
s3, freeze-drying the blending system I obtained in the step S2 to obtain a methylcellulose-collagen blending material II;
s4, taking 0.5 g of the blending material II obtained in the step S3, placing the blending material II in a beaker, adding 5 ml of deionized water, stirring at 15 ℃ (rotating speed of 100 r/min) for dispersion, placing the mixture in an ice water bath, stirring (100 r/min) for dissolution for 4 hours, and then refrigerating and stabilizing the mixture at 4 ℃ for 6 hours to obtain a methylcellulose-collagen solution;
s5, placing an air pump stone (with the diameter of 2cm) connected with an air pump device in the solution obtained in the step S4, pumping air at the air pumping speed of 10L/min, and after 1 minute, placing the solution in a water bath at 35 ℃ to prepare the methyl cellulose-collagen temperature-sensitive macroporous biological hydrogel.
Example 3
A preparation method of temperature-sensitive macroporous biological hydrogel comprises the following steps:
s1, weighing 1.5 g of methylcellulose (with the molecular weight of 10000 and the degree of substitution of 5.0), placing the methylcellulose in a beaker, adding 100 ml of ultrapure water, stirring at 60 ℃ (the rotation speed is 900 revolutions per minute) until the methylcellulose is completely dissolved, and cooling to room temperature to obtain a methylcellulose solution;
s2, weighing 0.1 g of type II collagen-bovine cartilage, adding the weighed type II collagen-bovine cartilage into the methylcellulose solution obtained in the step S1, stirring at 5 ℃ (900 revolutions per minute) for dissolving for 10 hours, and then refrigerating and stabilizing at 2 ℃ for 1 hour to obtain a methylcellulose-collagen blending system I;
s3, freeze-drying the blending system I obtained in the step S2 to obtain a methylcellulose-collagen blending material II;
s4, weighing 1.0 g of the blending material II obtained in the step S3, placing the blending material II in a beaker, adding 10 ml of ultrapure water, stirring at 50 ℃ (the rotating speed is 900 revolutions per minute) for dispersion, placing the mixture in an ice water bath, stirring (900 revolutions per minute) for dissolution for 8 hours, and then refrigerating and stabilizing the mixture at 6 ℃ for 6 hours to obtain a methylcellulose-collagen solution;
s5, placing an air pump stone (with the diameter of 5cm) connected with an air pump in the solution obtained in the step S4, pumping air at the air pumping speed of 0.1L/min, and after 10 minutes, placing the solution in a water bath at 40 ℃ to prepare the methyl cellulose-collagen temperature-sensitive macroporous biological hydrogel.
Example 4
A preparation method of temperature-sensitive macroporous biological hydrogel comprises the following steps:
s1, weighing 1.2 g of methylcellulose (with the molecular weight of 180000 and the substitution degree of 2.6), placing the methylcellulose in a beaker, adding 75 ml of normal saline, stirring at 50 ℃ (the rotation speed of 700 revolutions per minute) until the methylcellulose is completely dissolved, and cooling to room temperature to obtain a methylcellulose solution;
s2, weighing 0.08 g of type II collagen-bovine nasal membrane, adding the weighed type II collagen-bovine nasal membrane into the methylcellulose solution obtained in the step S1, stirring at 35 ℃ (700 revolutions per minute) for dissolving for 6 hours, and then refrigerating and stabilizing at 5 ℃ for 15 hours to obtain a methylcellulose-collagen blending system I;
s3, freeze-drying the blending system I obtained in the step S2 to obtain a methylcellulose-collagen blending material II;
s4, weighing 1.2 g of the blending material II obtained in the step S3, placing the blending material II in a beaker, adding 14 ml of normal saline, stirring at 42 ℃ (the rotating speed is 700 r/min), dispersing, placing the blending material II in an ice water bath, stirring (700 r/min), dissolving for 6 hours, and then refrigerating and stabilizing at 5 ℃ for 18 hours to obtain a methylcellulose-collagen solution;
s5, placing an air pump stone (the diameter is 3cm) connected with an air pump in the solution obtained in the step S4, pumping air, wherein the air pumping speed is 1.5L/min, and after 4 minutes, placing the solution in a water bath at 38 ℃ to prepare the methyl cellulose-collagen temperature-sensitive macroporous biological hydrogel.
Example 5
A preparation method of temperature-sensitive macroporous biological hydrogel comprises the following steps:
s1, weighing 0.8 g of methylcellulose (with the molecular weight of 100000 and the substitution degree of 4.8), placing the methylcellulose in a beaker, adding 90 ml of normal saline, stirring at 35 ℃ (the rotation speed is 500 r/min) until the methylcellulose is completely dissolved, and cooling to room temperature to obtain a methylcellulose solution;
s2, weighing 0.04 g of type II collagen-chicken, adding the chicken into the methylcellulose solution obtained in the step S1, stirring at 38 ℃ (500 rpm) to dissolve for 7 hours, and refrigerating and stabilizing at 4 ℃ for 25 hours to obtain a methylcellulose-collagen blending system I;
s3, freeze-drying the blending system I obtained in the step S2 to obtain a methylcellulose-collagen blending material II;
s4, weighing 0.8 g of the blending material II obtained in the step S3, placing the blending material II in a beaker, adding 8 ml of normal saline, stirring at 20 ℃ (the rotating speed is 500 r/min), dispersing, placing the blending material II in an ice water bath, stirring (500 r/min), dissolving for 7 hours, and then refrigerating and stabilizing at 5 ℃ for 18 hours to obtain a methylcellulose-collagen solution;
s5, placing an air pump stone (with the diameter of 4cm) connected with an air pump in the solution obtained in the step S4, pumping air at the air pumping speed of 1.2L/min, and after 3 minutes, placing the solution in a water bath at 39 ℃ to prepare the methyl cellulose-collagen temperature-sensitive macroporous biological hydrogel.
Example 6
The preparation method of the temperature-sensitive macroporous biological hydrogel of the present example is the same as that of example 1, except that steps S3 and S4 are not included, step S5 is directly performed, and the operation of preparing the hydrogel is performed by placing the air pump stone connected to the air pump device in the methylcellulose-collagen blending system I.
Comparative example 1
The preparation method of the comparative example is different from that of example 1 in that instead of using the emulsifier Span-80 to make holes, Span-80 is added into the methylcellulose-collagen blending system I, the mixture is stirred uniformly and then stands for 24 hours, and the gel is prepared after the Span-80 is washed away by water.
Comparative example 2
The preparation method of the comparative example is different from that of example 1 in that the hole making by using the air stone is replaced by the hole making by using the PVA hole making agent, Span-80 is added into the methylcellulose-collagen blending system I, the mixture is stirred uniformly and then stands for 24 hours, and the gel is prepared after the PVA is washed away by water.
Comparative example 3
The comparative example was prepared in the same manner as in example 1 except that the pumping speed was changed to 0.01L/min.
Comparative example 4
The comparative example was prepared in the same manner as in example 1 except that the pumping speed was changed to 15L/min.
Performance testing
As can be seen from fig. 2, when the methylcellulose-collagen solution in a liquid state at room temperature is pumped into air by the air pump stone, and is placed at 37 ℃ for 1 hour and then inverted, the methylcellulose-collagen solution becomes a non-flowing hydrogel at a temperature close to body temperature, which indicates that the temperature-sensitive macroporous hydrogel is prepared.
As can be seen from FIG. 3, after freeze-drying, the temperature-sensitive macroporous biological hydrogel has a loose hydrogel structure and a large number of pore structures, and the pore diameter can reach 200 μm, which proves that the prepared hydrogel has a macroporous structure.
The results of fig. 4 show that the temperature-sensitive macroporous bio-hydrogel prepared in example 1 has a storage modulus (G') higher than a loss modulus (G ") at a Strain (Strain) of less than 40%, indicating the characteristics of the hydrogel.
In addition, a surfactant or a pore-forming agent is not used in the process of preparing the hydrogel, so the prepared hydrogel is safe and non-toxic, does not generate toxic or side effect after long-term action, and can be widely applied to the biomedical fields of preparation of tissue administration, stem cell treatment, injection treatment preparations and the like. However, the steps of preparing the pores in comparative examples 1 and 2 both depend on a surfactant or a pore-forming agent, and although the gel is prepared and then removed by washing, no residue can be ensured, so that the application of the gel in the field of biomedicine is limited. In contrast, the air is introduced at a speed too low in comparative example 3, so that a macroporous structure cannot be formed, while the air is introduced at a speed too high in comparative example 4, so that the solution is blown up, and formed bubbles are particularly large and easy to burst, so that a large amount of uniform macroporous structures cannot be formed.
It should be understood that the above-described embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Claims (10)
1. A preparation method of temperature-sensitive macroporous biological hydrogel is characterized by dissolving methylcellulose and collagen in a solvent to obtain a methylcellulose-collagen mixed solution, introducing air at a speed of 0.1-10L/min, and preparing the temperature-sensitive macroporous biological hydrogel at 20-40 ℃, wherein the mass percentage concentration of the methylcellulose in the mixed solution is 8-15%, and the mass percentage concentration of the collagen in the mixed solution is 0.2-0.7%.
2. The method according to claim 1, wherein the air is introduced at a rate of 1.2 to 1.5L/min.
3. The method according to claim 1 or 2, wherein the methylcellulose has a molecular weight of 10000 to 180000 and a degree of methyl substitution of 2.6 to 5.0.
4. The method according to claim 1, wherein the collagen is one or more of type I collagen-bovine achilles tendon, type II collagen-bovine cartilage, type II collagen-bovine nasal membrane, and type II collagen-chicken.
5. The preparation method according to claim 1, wherein the methyl cellulose is dissolved in a solvent, heated and dissolved at 30-60 ℃, and cooled to room temperature to obtain a methyl cellulose solution; and adding the collagen into the methylcellulose solution, uniformly mixing at 5-40 ℃, refrigerating for 1-25 hours at 2-10 ℃ after 0.5-10 hours, and obtaining the methylcellulose-collagen mixed solution.
6. The method according to claim 1, wherein the methylcellulose-collagen mixture is freeze-dried to obtain a methylcellulose-collagen solid; dissolving the methylcellulose-collagen solid, and introducing air.
7. The preparation method according to claim 6, wherein the step of dissolving the methylcellulose-collagen solid comprises the steps of adding a solvent, dispersing at 15-50 ℃, dissolving in an ice-water bath for 4-8 hours, and refrigerating at 4-6 ℃ for 6-24 hours to obtain a solution with a mass concentration of 5-10%.
8. The preparation method according to claim 1, wherein the solution is placed in a water bath at 20-40 ℃ after air is introduced for 1-10 minutes to form the methylcellulose-collagen temperature-sensitive macroporous hydrogel.
9. The temperature-sensitive macroporous biological hydrogel prepared by the preparation method of the temperature-sensitive macroporous biological hydrogel according to any one of claims 1 to 8.
10. Use of the temperature-sensitive macroporous biological hydrogel of claim 9 in the preparation of a formulation for tissue administration, stem cell therapy and injectable therapy.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115998956A (en)* | 2023-01-17 | 2023-04-25 | 上海交通大学医学院附属第九人民医院 | Tissue regeneration method combining foaming hydrogel and cell microspheres |
| CN119097767A (en)* | 2024-08-26 | 2024-12-10 | 佛山市安珂捷生物科技有限责任公司 | A kind of acellular matrix hydrogel with adjustable stiffness and its preparation method and application |
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