CN114621465B - Collagen hydrogel and preparation method thereof - Google Patents
Collagen hydrogel and preparation method thereof Download PDFInfo
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- 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
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- 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
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
- C08K3/00—Use of inorganic substances as compounding ingredients
- C08K3/16—Halogen-containing compounds
- C08K2003/162—Calcium, strontium or barium halides, e.g. calcium, strontium or barium chloride
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
- C08K3/00—Use of inorganic substances as compounding ingredients
- C08K3/16—Halogen-containing compounds
- C08K2003/166—Magnesium halide, e.g. magnesium chloride
Abstract
The invention relates to a collagen hydrogel and a preparation method thereof, the collagen hydrogel of the invention respectively prepares a phase A and a phase B, the two phases are independently stored after being prepared, and are mixed before use, the preparation is carried out on site, the actual use requirement is better met, an acid solution is not needed to be added in the phase A, the use amount of a reagent and the experimental steps can be reduced, the hydrogel can be gelled under the condition of low temperature or room temperature, and the mechanical property is better; according to the invention, collagen is dissolved in a salt solution, so that more ammonium ions exist in the solution, a buffer solution system is added, the collagen is subjected to self-assembly, calcium ions or magnesium ions are divalent ions, and the swelling capacity of the divalent ions on the collagen is stronger, so that the ions in the buffer salt solution and a collagen chain are easier to react, and the self-assembly degree is higher.
Description
Technical Field
The invention belongs to the technical field of hydrogel, and particularly relates to a collagen hydrogel and a preparation method thereof.
Background
The core of tissue engineering is the construction of a cell and cell scaffold combined compound, and the three key elements of the tissue engineering are the formation and regeneration of a cell scaffold consisting of cells and biological materials and tissues and organs. The cell scaffold material made of the biomaterial has the characteristics of certain biodegradation speed, good biocompatibility and cell affinity, certain mechanical property, sterilization and disinfection and pore structure. Collagen is one of the main components of extracellular matrix, has the advantages of unique physicochemical property, excellent biocompatibility, degradability, low immunity and the like, is easy to absorb in organism, has strong hydrophilicity, no toxicity and good safety, and becomes one of the choices of biomedical materials. Molecular self-assembly refers to the process by which molecules spontaneously form a stable, ordered supramolecular aggregate or supramolecular structure under certain equilibrium conditions through the action of non-covalent bonds. Collagen has self-assembly property, in vivo, the head and tail of the procollagen are staggered by one quarter and self-assembled, and are crosslinked through covalent bonds to form a fiber structure with D-band characteristics, and further aggregated into bundles to form collagen fibers, so as to build various tissues in the human body. The conditions affecting collagen self-assembly include collagen concentration, temperature, pH, solution concentration, and solution ion species. The collagen hydrogel prepared in the prior art has low gelling speed, can be gelled only by slowly mixing additives, can generate temperature change when being used for superficial tissues of a human body, and is reversible and not suitable for the human body.
The present invention has been made in view of the above circumstances.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides the collagen hydrogel and the preparation method thereof, the hydrogel prepared by the invention has short gelling time, can be mixed at low temperature and normal temperature, has good mechanical property, and can be mixed with the phase A and the phase B before use, thereby being more in line with the actual use requirements.
The first object of the present invention is to provide a method for preparing a collagen hydrogel, comprising the steps of:
(1) preparation of phase A: adding collagen into the calcium chloride solution or the magnesium chloride solution, and fully mixing to obtain a collagen solution;
(2) preparation of phase B: preparing a buffer solution system, wherein the buffer solution system is one of phosphate buffer solution, Tris-HCl buffer solution and barbital sodium-hydrochloric acid buffer solution;
(3) and respectively filling the phase A and the phase B into different containers, and mixing the phase A and the phase B when in use to obtain the collagen hydrogel.
Further, the concentration of calcium chloride in the calcium chloride solution in the step (1) is 125-350 mM.
Further, the concentration of calcium chloride in the calcium chloride solution was 240 mM.
Further, the concentration of magnesium chloride in the magnesium chloride solution in the step (1) is 100-300 mM.
Further, the mass fraction of the collagen in the step (1) is 0.6-4.5% of that of the calcium chloride solution or the magnesium chloride solution.
Further, the phosphate buffer solution in the step (2) is prepared by mixing disodium hydrogen phosphate and potassium dihydrogen phosphate according to a molar ratio of (10: 1) - (5: 1), adding water until the concentration of total phosphate is 0.04-0.06M, and adjusting the pH value to 7-8 by using acid and alkali.
Further, the Tris-HCl buffer solution in the step (2) is prepared by mixing Tris (hydroxymethyl) aminomethane and hydrochloric acid according to a molar ratio of (3-5) to (3-5), adding water until the total concentration of Tris (hydroxymethyl) aminomethane and hydrochloric acid is 0.275-0.525M, and adjusting the pH value to 7.2-7.6 by using acid and alkali.
The pH value of the buffer solution is related to the proportion of the raw materials, and the pH value is adjusted to reach the required range through the acid and the alkali together according to the measured pH value before the pH value is adjusted.
Further, the barbiturate sodium-hydrochloric acid buffer solution in the step (2) is prepared by mixing barbiturate sodium and hydrochloric acid according to a molar ratio of (5-8) to (4-7), adding water until the total concentration of the barbiturate sodium and the hydrochloric acid is 0.175-0.315M, and adjusting the pH value to 7.2-7.6 by using acid and alkali.
Further, the mixing volume ratio of the phase A and the phase B in the step (3) is (2: 1) - (4: 1).
Further, the mixing temperature is 5-25 deg.C, and the use temperature is 5-37 deg.C.
In a second object of the present invention, there is provided a collagen hydrogel prepared by the method.
The collagen hydrogel disclosed by the invention has self-assembly property based on the collagen, the self-assembly rate, time and self-assembly capacity can be influenced by changing factors such as collagen concentration, ion concentration in a solution, pH value and temperature, and the collagen solution can be gradually converted into a gel state from a liquid state under a proper condition. The proportion and the conditions of the raw materials in the phase A and the phase B are obtained by a large number of experiments, the hydrogel prepared under the conditions of the invention can be gelled at low temperature or room temperature, and the mechanical property is better.
The phase A and the phase B are independently stored after being prepared and are mixed before use.
Compared with the prior art, the invention has the following beneficial effects:
(1) the collagen hydrogel is prepared into a phase A and a phase B respectively, the two phases are independently stored after being prepared, and are mixed before use, the collagen hydrogel is prepared at present and better meets the actual use requirement, an acid solution is not required to be added into the phase A, the use amount of a reagent and the experimental steps can be reduced, the collagen hydrogel can be formed into gel at low temperature or room temperature, and the mechanical property is better;
(2) according to the invention, the collagen is dissolved in the salt solution, so that more ammonium ions exist in the solution, and then the buffer solution system is added, so that the collagen is subjected to self-assembly, and the calcium ions or the magnesium ions are divalent ions, and the swelling capacity of the divalent ions on the collagen is known from the influence of various ions on the swelling capacity of the collagen, so that the ions in the buffer salt solution and the collagen chains are easier to react, and the self-assembly degree is higher.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
Example 1
The preparation method of the collagen hydrogel of the embodiment comprises the following steps:
(1) preparation of phase A: adding 2% collagen into 300mM calcium chloride solution, and mixing completely to obtain collagen solution, i.e. phase A;
(2) preparation of phase B: mixing disodium hydrogen phosphate and potassium dihydrogen phosphate at a molar ratio of 5:1, adding water to a total phosphate concentration of 0.04M, and adjusting pH to 7-8 with hydrochloric acid and sodium hydroxide to obtain phosphate buffer solution as phase B.
The collagen hydrogel prepared in this example was used with phase a and phase B mixed in a volume ratio of 4: 1.
Example 2
The preparation method of the collagen hydrogel of the embodiment comprises the following steps:
(1) preparation of phase A: adding 0.6% collagen into 125mM calcium chloride solution, and mixing well to obtain collagen solution, i.e. phase A;
(2) preparing a phase B: mixing disodium hydrogen phosphate and potassium dihydrogen phosphate at a molar ratio of 10:1, adding water until the total phosphate concentration is 0.05M, and adjusting pH to 7-8 with hydrochloric acid and sodium hydroxide to obtain phosphate buffer solution, i.e. phase B.
The collagen hydrogel prepared in this example was used by mixing phase A and phase B at a volume ratio of 2: 1.
Example 3
The preparation method of the collagen hydrogel of the embodiment comprises the following steps:
(1) preparation of phase A: adding 3% of collagen into 350mM calcium chloride solution, and fully and uniformly mixing to obtain collagen solution, namely phase A;
(2) preparation of phase B: mixing disodium hydrogen phosphate and potassium dihydrogen phosphate at a molar ratio of 7.5:1, adding water to a total phosphate concentration of 0.06M, and adjusting pH to 7-8 with hydrochloric acid and sodium hydroxide to obtain phosphate buffer solution, i.e. phase B.
The collagen hydrogel prepared in this example was used by mixing phase a and phase B at a volume ratio of 3: 1.
Example 4
The preparation method of the collagen hydrogel of the embodiment comprises the following steps:
(1) preparation of phase A: adding 2% collagen into 100mM magnesium chloride solution, and mixing to obtain collagen solution A phase;
(2) preparing a phase B: mixing the trihydroxymethyl aminomethane and hydrochloric acid according to a molar ratio of 3:5, adding water until the total concentration of the trihydroxymethyl aminomethane and the hydrochloric acid is 0.275M, and adjusting the pH value to 7.2-7.6 by using the hydrochloric acid and sodium hydroxide to obtain a phase B.
The collagen hydrogel prepared in this example was used by mixing phase A and phase B at a volume ratio of 2: 1.
Example 5
The preparation method of the collagen hydrogel of the embodiment comprises the following steps:
(1) preparation of phase A: adding 0.5% collagen into 200mM magnesium chloride solution, and mixing well to obtain collagen solution, i.e. phase A;
(2) preparation of phase B: mixing the trihydroxymethyl aminomethane and hydrochloric acid according to the molar ratio of 1:1, adding water until the total concentration of the trihydroxymethyl aminomethane and hydrochloric acid is 0.4M, and adjusting pH value to 7.2-7.6 with hydrochloric acid and sodium hydroxide to obtain phase B. The collagen hydrogel prepared in this example was used by mixing phase A and phase B at a volume ratio of 3: 1.
Example 6
The preparation method of the collagen hydrogel of the embodiment comprises the following steps:
(1) preparation of phase A: adding 3% of collagen into 300mM magnesium chloride solution, and fully and uniformly mixing to obtain collagen solution, namely phase A;
(2) preparation of phase B: mixing the trihydroxymethyl aminomethane and hydrochloric acid according to the molar ratio of 5:3, adding water until the total concentration of the trihydroxymethyl aminomethane and hydrochloric acid is 0.525M, and adjusting the pH value to 7.2-7.6 with hydrochloric acid and sodium hydroxide to obtain phase B. The collagen hydrogel prepared in this example was used with phase a and phase B mixed in a volume ratio of 4: 1.
Example 7
The preparation method of the collagen hydrogel of the embodiment comprises the following steps:
(1) preparation of phase A: adding 2% collagen into 300mM calcium chloride solution, and mixing completely to obtain collagen solution, i.e. phase A;
(2) preparation of phase B: mixing barbital sodium and hydrochloric acid at a molar ratio of 5:7, adding water until the total concentration of barbital sodium and hydrochloric acid is 0.175M, and adjusting pH to 7.2-7.6 with hydrochloric acid and sodium hydroxide to obtain phase B.
The collagen hydrogel prepared in this example was used by mixing phase a and phase B at a volume ratio of 2: 1.
Example 8
The preparation method of the collagen hydrogel of the embodiment comprises the following steps:
(1) preparation of phase A: adding 4.5% collagen into 100mM magnesium chloride solution, and mixing to obtain collagen solution A phase;
(2) preparation of phase B: mixing barbiturate sodium and hydrochloric acid at a molar ratio of 7:5, adding water until the total concentration of barbiturate sodium and hydrochloric acid is 0.245M, and adjusting pH to 7.2-7.6 with hydrochloric acid and sodium hydroxide to obtain phase B.
The collagen hydrogel prepared in this example was used by mixing phase a and phase B at a volume ratio of 3: 1.
Example 9
The preparation method of the collagen hydrogel of the embodiment comprises the following steps:
(1) preparation of phase A: adding 3% of collagen into 350mM calcium chloride solution, and fully and uniformly mixing to obtain collagen solution, namely phase A;
(2) preparation of phase B: mixing barbital sodium and hydrochloric acid at a molar ratio of 8:4, adding water until the total concentration of barbital sodium and hydrochloric acid is 0.315M, and adjusting pH to 7.2-7.6 with hydrochloric acid and sodium hydroxide to obtain phase B.
The collagen hydrogel prepared in this example was used with phase a and phase B mixed in a volume ratio of 4: 1.
Comparative example 1
The preparation method of the collagen hydrogel of the comparative example comprises the following steps:
(1) preparation of phase A: dissolving 2.0% collagen in 0.01M hydrochloric acid at 4 deg.C to obtain collagen solution, and storing at 4 deg.C;
(2) preparing a phase B: preparing a buffer solution, wherein the buffer solution contains 680mM Na+Solution, 60mM HPO4 2-/H2PO4 -The solution was slowly added with 0.05M sodium hydroxide solution, the pH of the solution was adjusted to 7.4, and placed in a water bath at different temperatures (5 ℃ C., 37 ℃ C.).
The collagen hydrogel prepared in this example was used with phase a and phase B mixed in a volume ratio of 4: 1.
Comparative example 2
The preparation method of the collagen hydrogel of the comparative example comprises the following steps:
(1) preparation of phase A: dissolving 2.0% collagen in hydrochloric acid solution with pH of 2.5 to obtain collagen solution;
(2) preparing a phase B: a buffer solution of pH 7.4 containing 680mM Na was prepared+Solution, 60mM HPO4 2-/H2PO4 -The solution of (4) is placed in a water bath at 25 ℃.
The collagen hydrogel prepared in this example was used by mixing phase a and phase B at a volume ratio of 4: 1.
The gel formation times in the following test examples were all observed once for 5s and recorded.
Test example 1
The hydrogels prepared in example 1 and comparative examples 1-2 were tested for gel formation time and maximum storage modulus at different mixing and use temperatures.
The self-assembly capacity of the collagen solution is represented by measuring the storage modulus and the loss modulus through rheological properties, the mixed sample is detected at a fixed frequency in an environment of 37 ℃, points are taken once every 5s within 300s, 60 measurement points are counted, the storage modulus is recorded, the first time point when the storage modulus reaches the maximum value of the storage modulus, namely 90%, is taken as the gelling time, and the result is shown in table 1.
As can be seen from Table 1, the hydrogel prepared by the invention can be gelled under the conditions of low temperature or normal temperature, low temperature, normal temperature and body temperature, and has certain mechanical properties.
The above experiments were also carried out by the present inventors on hydrogels prepared in other examples, and the results were substantially consistent and not listed again due to space limitation.
Test example 2
The phase a collagen solutions prepared in examples 1 to 9 and comparative examples 1 to 2 were subjected to thermal denaturation temperature analysis, and the peak value of the endothermic peak of collagen denaturation was defined as a thermal denaturation temperature, the peak area integral was an enthalpy change value, and the enthalpy change value was an inherent property of the test material, and the results were as shown in table 2.
As can be seen from the data in Table 2, the hydrogels prepared in the examples of the present invention and the comparative examples have no significant difference in heat denaturation temperature and enthalpy change value, indicating that the method of the present invention does not change the heat denaturation temperature of the hydrogel.
Test example 3
The hydrogels prepared in examples 1-9 and comparative examples 1-2 were tested for degradation testing as follows: the mixing temperature was 25 ℃ and gelling was carried out at the use temperature of 37 ℃ and 1500mg of each of examples 1 to 9 and comparative examples 1 to 2 was weighed and immersed in 100mL of 5U/mL collagenase PBS buffer solution at 37 ℃ to evaluate the degradation time for 24 hours, and the degree of degradation of the sample was evaluated by estimating the remaining amount of the sample by measuring the content of hydroxyproline in the solution, as shown in Table 3.
As can be seen from the data in Table 3, the more hydroxyproline content in the solution, the less sample remaining, demonstrating faster degradation. Within 24h of degradation, the hydroxyproline content of the invention is less than that of comparative examples 1-2. The examples 1 to 9 all formed gel, and the surface area of the gel-formed sample contacted with the collagenase solution was decreased, while the comparative example 1 did not form gel, and the sample was dispersed, and the surface area of the gel-formed sample contacted with the collagenase solution was increased, so that the degradation was faster, and the hydroxyproline content in the solution was higher, and the results showed that the hydrogel prepared by the present invention had longer degradation time than those of comparative examples 1 to 2, so that the method of the present invention promoted the self-assembly of collagen, and the hydrogel prepared by the method of the present invention had strong gelling ability.
Test example 4
The phases A and B prepared in examples 1-9 were mixed together at 25 ℃ and directly injected into PBS solution at 37 ℃ to form a whole colloid in a short time, while comparative examples 1-2 were relatively dispersed and could not be formed into a colloid. Indicating that the hydrogel of the invention can gel in an aqueous environment.
Test example 5
The hydrogel was prepared according to the method of example 1, only by changing the amount of collagen added, the mixing temperature was 25 ℃, the use temperature was 37 ℃, the effect of different collagen contents on the properties of the prepared hydrogel was studied, the specific test methods were the same as those of experimental example 1 and experimental example 3, and the results are shown in table 4.
As can be seen from the results in table 4, as the concentration of collagen increases, the more molecules in the unit space, the faster the collagen molecules are aggregated together, the faster the self-assembly degree and time are, the shorter the gelling time, the longer the degradation time of the formed gel, and when the percentage of collagen is greater than 5.0%, the longer the gelling time is, because the concentration of collagen is high, there is more competitiveness in the process of mixing with calcium chloride or magnesium chloride solution to form ammonium ions. Meanwhile, the collagen has high concentration and is mixed with the buffer solution to cause high viscosity, so that the collagen is not uniformly contacted with the buffer solution, and the time required by gelling is prolonged.
Test example 6
The hydrogel was prepared according to the method of example 1, and the influence of the properties of the hydrogel prepared at different collagen contents was investigated by changing the concentration of the calcium chloride solution only at a mixing temperature of 25 ℃ and a use temperature of 37 ℃, and the specific test methods were the same as those of test examples 1 and 3, and the results are shown in table 5.
From the results in Table 5, it can be seen that as the ion concentration increases, the ions in the solution are more easily combined with the water molecules, more collagen molecules are exposed for self-assembly, and the degradation time is longer, but when the ion concentration exceeds a certain range (350 mM), the collagen is salted out, the gelling time is increased, and the self-assembly process is inhibited.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Claims (6)
1. A method for preparing a collagen hydrogel, comprising the steps of:
(1) preparation of phase A: adding collagen into the calcium chloride solution or the magnesium chloride solution, and fully mixing to obtain a collagen solution, wherein the concentration of calcium chloride in the calcium chloride solution is 125-350mM, the concentration of magnesium chloride in the magnesium chloride solution is 100-300mM, and the mass fraction of the collagen is 0.6-4.5% of that of the calcium chloride solution or the magnesium chloride solution;
(2) preparation of phase B: preparing a buffer solution system, wherein the buffer solution system is one of phosphate buffer solution, Tris-HCl buffer solution and barbital sodium-hydrochloric acid buffer solution;
(3) and respectively filling the phase A and the phase B into different containers, and mixing the phase A and the phase B when in use, wherein the mixing volume ratio of the phase A to the phase B is (2: 1) - (4: 1), so as to obtain the collagen hydrogel.
2. The method for preparing a collagen hydrogel according to claim 1, wherein said phosphate buffer solution in step (2) is prepared by mixing disodium hydrogenphosphate and potassium dihydrogenphosphate at a molar ratio of (10: 1) - (5: 1), adding water to a total phosphate concentration of 0.04-0.06M, and adjusting pH to 7-8 with an acid and a base.
3. The method for preparing a collagen hydrogel according to claim 1, wherein said Tris-HCl buffer solution of the step (2) is prepared by mixing Tris and hydrochloric acid at a molar ratio of (3-5) to (3-5), adding water to a total concentration of Tris and hydrochloric acid of 0.275-0.525M, and adjusting pH to 7.2-7.6 with an acid and a base.
4. The method for preparing a collagen hydrogel according to claim 1, wherein said barbital sodium-hydrochloric acid buffer solution in step (2) is prepared by mixing barbital sodium and hydrochloric acid in a molar ratio of (5-8) to (4-7), adding water to a total concentration of barbital sodium and hydrochloric acid of 0.175-0.315M, and adjusting pH to 7.2-7.6 with an acid and a base.
5. The method for preparing a collagen hydrogel according to claim 1, wherein the mixing temperature in the step (3) is 5 to 25 ℃ and the use temperature is 5 to 37 ℃.
6. A collagen hydrogel produced by the method of any one of claims 1 to 5.
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| CN110698866A (en) * | 2019-10-17 | 2020-01-17 | 四川大学 | Ultrasonic-mediated silk fibroin composite collagen hydrogel and preparation method thereof |
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