CN110746516B - Natural polymer hydrogel based on collagen and preparation method thereof - Google Patents
Natural polymer hydrogel based on collagen and preparation method thereof Download PDFInfo
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- CN110746516B CN110746516B CN201911035845.3A CN201911035845A CN110746516B CN 110746516 B CN110746516 B CN 110746516B CN 201911035845 A CN201911035845 A CN 201911035845A CN 110746516 B CN110746516 B CN 110746516B
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- glycogen
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Abstract
The invention discloses a natural polymer hydrogel based on collagen and a preparation method thereof, belonging to the field of hydrogels. The invention firstly synthesizes a glycogen derivative used as cross-linking agent guanidino glycogen oxide, the glycogen derivative has a plurality of groups on the surface, inorganic component hydroxyapatite and organic component collagen can be connected through chemical bonds and electrostatic action, natural polymer materials and inorganic materials can be combined and used together, and thereby, the hydrogel which can effectively and directionally induce the differentiation of MSCs is formed. The natural polymer hydrogel material based on collagen prepared by the invention has certain stretchability, elasticity and bendability, the compression modulus and the elastic modulus are in the range of the interval capable of differentiating the bone marrow mesenchymal stem cells into cartilage and osteogenesis, and the natural polymer hydrogel material can be ensured not to be easily damaged when large bone injury is faced.
Description
Technical Field
The invention relates to a natural polymer hydrogel based on collagen and a preparation method thereof, belonging to the field of hydrogels.
Background
People in modern society have a fast pace of life and are accustomed to fast walking, so that bone and cartilage injuries are easily caused. Because the bone tissue lacks lymph, blood vessels and nerve distribution, and lacks nutrition supply in the repair process, the bone tissue cannot be effectively self-repaired only by limited nutrition of stroma and joint cavity synovial fluid and weak self-division capability of osteogenesis and cartilage cells. After cartilage injury, clinical symptoms such as knee joint pain, joint instability, joint stiffness and the like can be caused, secondary osteoarthritis can occur in severe cases, the secondary osteoarthritis is also an important reason for limb dysfunction and disability, and the life quality of patients can be seriously affected. The repair of cartilage damage is a major concern in modern medicine.
The existing bone injury treatment methods are mainly divided into two methods, namely an operation method and a tissue engineering method. Although the operation method has a certain curative effect, the effect of improving the knee joint function of a patient is limited, the operation process is complex, the operation difficulty is high, and the patient still needs to endure the pain caused by the operation. Tissue engineering can alter the differentiation of stem cells by mimicking key parts of the extracellular environment, wherein Mesenchymal Stem Cells (MSCs) are pluripotent cells that can produce mesenchymal and non-mesenchymal tissues in vitro and in vivo, and have excellent differentiation ability, differentiating into osteoblasts, chondrocytes, and adipocytes. Furthermore, the differentiation of stem cells can also be regulated by modulating the physical properties of the biomaterial, including the composition, mechanical properties, 2D morphology and 3D space, among other factors, especially the specific properties of the chemical function on the 2D surface such as stiffness, surface charge and hydrophilicity, which have been shown to have a significant impact on the differentiation of stem cells. Extracellular matrix (ECM) -derived proteins and/or peptides, such as collagen type I, are introduced onto a matrix for selectively directing neurogenesis, myogenesis and osteogenesis. In addition to organic molecules, inorganic elements can also be used to direct cell differentiation, for example, the prior art has found that adding a hydroxyapatite phase to a material has better cell and protein adhesion, enhanced cell proliferation and higher osteogenic gene expression.
In the aspect of cartilage and osteogenic repair, related researches on biological scaffolds and sponges are performed domestically and internationally. The biological scaffold and sponge can be used as filler and guide the adhesion growth of stem cells on the biological scaffold and sponge. Compared with solid scaffolds such as scaffolds and sponges, the hydrogel material has a three-dimensional network structure which is more hydrophilic, can rapidly swell in water and can retain a large volume of water without dissolving. The hydrogel has high value in the application aspect of tissue engineering, and after the hydrogel is implanted into a living body, the contact surface between the hydrogel and biological fluid and tissue tends to be fuzzy, so that the surface tension can be reduced, the adsorption of cells and proteins on the surface can be reduced, the friction between the hydrogel and the surrounding can be reduced, the damage to the surrounding tissue can be reduced, and the inflammation can be avoided.
At present, the prior art discloses a report of preparing a cartilage material by combining collagen and hydroxyapatite, but the prior art mainly mixes the collagen and the collagen simply and physically, the hydroxyapatite and the collagen cannot be crosslinked by the prior art, the collagen and the hydroxyapatite are very easy to separate due to different densities and existing forms in vivo in the subsequent use process, and certain risks exist in vivo application, so that a hydrogel system which can overcome the problem that the collagen and the hydroxyapatite cannot coexist for a long time needs to be found.
Disclosure of Invention
[ problem ] to provide a method for producing a semiconductor device
The hydrogel in the prior art has the problems that inorganic components are not uniformly distributed in the hydrogel and cannot be stabilized for a long time.
[ technical solution ] A
In order to solve the problems, the invention firstly synthesizes a glycogen derivative used as a cross-linking agent, the glycogen derivative has a plurality of groups on the surface, inorganic component hydroxyapatite and organic component collagen can be connected through chemical bonds and electrostatic action, and natural polymer materials and inorganic materials can be combined and used together, so that the hydrogel capable of effectively and directionally inducing the differentiation of the MSCs is formed.
Firstly, the invention provides guanidino glycogen oxide, wherein the guanidino glycogen oxide has a structural formula as follows:
wherein m is more than or equal to 1, n is more than or equal to 1, i is more than or equal to 1, and the average molecular weight is 8000-1000 kDa.
Secondly, the invention provides a preparation method of the guanidino glycogen oxide, which comprises the following steps:
(1) electropositivity of glycogen: dissolving glycogen in dimethyl sulfoxide to form a solution with the mass-to-volume ratio of 0.5-2% (the unit is g/mL, the same below), then adding N, N-carbonyl diimidazole with the amount being 1-20 times that of the glycogen, stirring for 1 hour under the protection of inert atmosphere, then dissolving ethylenediamine with the amount being 5-50 times that of the glycogen in 5mL of dimethyl sulfoxide to obtain an ethylenediamine solution, dropwise adding the ethylenediamine solution into the activated reaction solution, reacting for 12-24 hours under the protection of inert atmosphere, and purifying and drying to obtain aminated glycogen;
(2) grafting a guanidino group on aminated glycogen: dissolving guanidinoacetic acid in water to obtain a guanidinoacetic acid solution with the mass-to-volume ratio of 0.5-2% and adjusting the pH to 5-6, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride in an amount which is 1-5 times of the amount of the guanidinoacetic acid and N-hydroxysuccinimide in an amount which is 1-5 times of the amount of the guanidinoacetic acid into the guanidinoacetic acid solution, activating for 0.5-4 hours, adding aminated glycogen prepared in the step (1) in an amount which is 0.1-1 times of the amount of the guanidinoacetic acid and adjusting the pH of the solution to 7-8, reacting for 12-16 hours, and purifying and drying to obtain guanidinoglycogen;
(3) oxidation of guanidino glycogen: and (3) dissolving a proper amount of the guanidino glycogen prepared in the step (2) in water to obtain a guanidino glycogen solution with the mass-volume ratio of 0.5-2%, adding sodium periodate with the amount of 1-5 times that of the guanidino glycogen, carrying out light-shielding reaction for 1-3 hours, and purifying and drying to obtain the guanidino glycogen oxide.
In one embodiment of the invention, the purification is carried out by dialyzing the product against 3500Da, 7000Da or 14000Da dialysis bags for 24-72 h.
In one embodiment of the invention, the drying is preferably freeze drying.
In one embodiment of the present invention, the method for adjusting pH in step (2) is adjusting with hydrochloric acid or sodium hydroxide, and the concentration of the hydrochloric acid or sodium hydroxide is preferably 1 mol/L.
In one embodiment of the present invention, in the step (3), at the end of the reaction, ethylene glycol is added in an amount of 2 to 10 times that of the substance of guanidino glycogen to terminate the reaction.
In one embodiment of the present invention, the inert atmosphere is nitrogen, helium, argon, or the like.
Third, the present invention provides a method for preparing a collagen-based natural polymer hydrogel, comprising: respectively preparing a collagen water solution with the concentration of 11.25-22.5mg/mL and a guanidino glycogen oxide solution with the concentration of 37.5-75mg/mL, and then adding 0-25mg of nano-hydroxyapatite into each 1mL of the guanidino glycogen oxide solution to obtain a guanidino glycogen oxide solution doped with the nano-hydroxyapatite; mixing a collagen water solution and a guanidino glycogen oxide solution doped with nano-hydroxyapatite according to a volume ratio of 2:1, then dripping a carbonate buffer solution on the mixed solution after 15-60 minutes, standing for 2-24 hours, reducing for 0.5-2 hours by using a sodium borohydride solution with the mass volume fraction of 0.05% after being taken out from a mould, washing with water, and sterilizing (such as ultraviolet sterilization) to obtain the collagen-based natural polymer hydrogel.
In one embodiment of the present invention, the number of washing with water is 1 to 6; the sterilization is carried out by irradiating under an ultraviolet lamp for 0.5-4 h.
In one embodiment of the present invention, the carbonate buffer is a sodium carbonate-sodium bicarbonate solution with a concentration of 0.5-2 mol/L.
In one embodiment of the present invention, the water is preferably ultrapure water.
Fourthly, the invention provides the natural polymer hydrogel based on collagen prepared by the preparation method.
Fifth, the present invention provides a health product or a medicine comprising the above collagen-based natural polymer hydrogel.
Sixth, the present invention provides the use of the above collagen-based natural polymer hydrogel in the differentiation of MSCs.
The beneficial technical effects obtained by the invention are as follows:
(1) in the natural polymer hydrogel based on collagen prepared by the invention, collagen is crosslinked through guanidino glycogen oxide, nano-hydroxyapatite is uniformly dispersed in the guanidino glycogen oxide, a light yellow solid gel is finally formed, the gel has certain stretchability, elasticity and bendability, and the compression modulus and the elastic modulus are in a range of a region capable of enabling bone marrow mesenchymal stem cells to be differentiated into cartilage and bone (the cartilage differentiation compression modulus is 80kPa, the bone differentiation compression modulus is 190kPa, and the bone differentiation elastic modulus is 40-80 kPa). Has proliferation promoting effect on fibroblast growth.
(2) In the natural polymer hydrogel based on collagen, because the collagen has excellent biocompatibility, flexibility and degradability, and the glycogen has excellent biocompatibility, water absorption capacity and water retention capacity, the formed hydrogel can provide an environment which is easy to adhere, proliferate and differentiate for cells, and is beneficial to the growth and differentiation of stem cells; the addition of the hydroxyapatite better simulates the bone environment, and simultaneously, the mechanical strength of the hydrogel is also obviously improved, and the finally synthesized natural high-molecular hydrogel material based on collagen is not easy to be damaged when facing larger bone injury, thereby greatly prolonging the service life of the hydrogel material.
Drawings
FIG. 1 is a graph showing the relationship between the volume of the added sodium hydroxide solution and the conductivity obtained when the content of amino groups in the aminated glycogen is measured by conductometry, and a schematic diagram showing the determination methods of V1 and V2.
FIG. 2 is a schematic diagram of a synthetic route to guanidino-oxide glycogen, wherein CDI represents N, N-carbonyldiimidazole; EDC represents 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride; NHS represents N-hydroxysuccinimide.
FIG. 3 1H-NMR spectra of glycogen, aminated glycogen, guanidino glycogen and guanidino glycogen oxide samples.
FIG. 4 results of potential measurement of glycogen, aminated glycogen, guanidino glycogen and guanidino-oxidized glycogen particles.
FIG. 5 shows the particle size distribution results of guanidino glycogen oxide doped with nano-hydroxyapatite obtained by mixing guanidino glycogen oxide and nano-hydroxyapatite in different proportions.
FIG. 6 is a TEM image of guanidino glycogen doped with nano-hydroxyapatite after mixing the guanidino glycogen with the nano-hydroxyapatite at a mass ratio of 10: 1.
FIG. 7 compressive modulus data of collagen-based natural polymer hydrogels prepared in examples 2-11.
FIG. 8 data from a cyclic compression experiment of the hydrogel prepared in example 9.
FIG. 9 is a graph showing cytotoxicity data of collagen-based natural polymer hydrogels prepared in examples 2 to 11 against 3T3 fibroblasts.
FIG. 10 experimental data for determination of alkaline phosphatase (ALP) content in mesenchymal stem cells grown on collagen-based natural polymer hydrogel prepared as described in examples 3, 4, 5, 7, 8, 9, 10, 11.
Detailed Description
Collagen is available from Wuxi Bedy bioengineering GmbH (soluble collagen powder).
Determination of amino content in aminated glycogen by conductivity titration method: weighing a proper amount of aminated glycogen to be dissolved in deionized water, adding 2mol/L hydrochloric acid to adjust the pH value to 2.0, titrating by using a calibrated 0.1mol/L sodium hydroxide solution, measuring the conductivity by using a conductivity meter to obtain a relation graph (shown in figure 1) between the addition volume of the sodium hydroxide solution and the conductivity, respectively making three slope lines of which the conductivity changes along with the volume of the added NaOH according to the trend, intersecting the three slope lines to obtain two intersection points, wherein the volumes of NaOH corresponding to the two intersection points are V from small to large1、V2The amino group content can be calculated by the following formula:
wherein, CNaOHThe concentration of NaOH is expressed in mol/L; mNH2Represents the molar mass of the amino groups in g/mol; wDry weight of test specimenRepresents the mass of the weighed aminated glycogen, and the unit is g; v1, V2 indicate the abscissa, i.e. volume, at the intersection in L.
Measuring the content of guanidino in guanidino glycogen by utilizing sakakou reaction: preparing guanidinoacetic acid standard solutions with different concentrations, 14 percent sodium hydroxide solution and color developing solution (40mg/ml alpha-naphthol, 0.2mg/ml diacetyl and n-propanol as a solvent). A certain amount of guanidino glycogen is dissolved in deionized water. Adding 3ml of guanidinoacetic acid solution and guanidino glycogen solution with different concentration gradients into 0.5ml of sodium hydroxide solution, 1.5ml of color development liquid and 1ml of n-propanol respectively, mixing uniformly, placing in a water bath kettle at 40 ℃ for 10min, cooling with tap water, placing at room temperature and keeping out of the sun. Measuring the absorbance of each solution at 530nm wavelength by using an ultraviolet spectrophotometer, and drawing a guanidinoacetic acid concentration-absorbance standard curve (R)2>0.999), then measuring the absorbance of the guanidino glycogen solution, and substituting the absorbance of the guanidino glycogen into a standard curve to obtain the guanidino content of the guanidino glycogen.
And (3) measuring the content of aldehyde group in the guanidino glycogen by a titration method: and (3) preparing a hydroxylamine hydrochloride solution with the pH value of 4, dissolving a proper amount of guanidino glycogen oxide in 25mL of hydroxylamine hydrochloride solution, and reacting for 2 hours at room temperature on a magnetic stirrer. After the reaction is finished, the solution is titrated by a calibrated 0.1M NaOH solution, and the change condition of the pH of the solution is monitored in real time by a pH meter. Until its pH was restored to 4, the volume of NaOH solution used was recorded. The aldehyde group content can be calculated by the following formula:
wherein, CNaOHThe concentration of NaOH is expressed in mol/L; mCHORepresents the molar mass of aldehyde groups, and the unit is g/mol; wDry weight of test specimenRepresents the mass of the weighed aminated glycogen, and the unit is g; v represents the volume of NaOH consumed in L.
The charged condition of the particles is measured by a potentiometer, and the specific method comprises the following steps: the charging of the solution of four particles of glycogen, aminated glycogen, guanidino glycogen and guanidino glycogen oxide was determined using a ZEN3600 Malvern particle size potentiometer. The four particles are respectively dissolved by deionized water to prepare 1mg/mL, and the solution is filled in a particle size cup for measurement.
Example 1
Guanidino-oxide glycogen and its preparation method (scheme of guanidino-oxide glycogen is shown in figure 2):
(1) electropositivity of glycogen: weighing 1g of glycogen to be dissolved in 70mL of anhydrous dimethyl sulfoxide in a three-neck flask, adding 1g N, N-carbonyl diimidazole after complete dissolution, activating for 1 hour under the protection of nitrogen, then dissolving 1.54mL of ethylenediamine in 10mL of dimethyl sulfoxide, slowly dripping into a reaction by using an injector, reacting for 24 hours under the protection of nitrogen, dialyzing a product in a 3500Da dialysis bag for 48 hours, and freeze-drying to obtain the aminated glycogen;
(2) grafting a guanidino group on aminated glycogen: weighing 537.5mg of guanidinoacetic acid, dissolving in 50ml of deionized water, adjusting the pH to 5.5 by using 1mol/L hydrochloric acid or sodium hydroxide, adding 1.32g of carbodiimide hydrochloride and 792.5mg of N-hydroxysuccinimide into the solution, adding 1g of aminated glycogen obtained in the step (1) after activating for 2 hours, adjusting the pH of the solution to 7.5 by using 1mol/L hydrochloric acid or sodium hydroxide, reacting for 16 hours, dialyzing the product in a 3500 dialysis bag for 48 hours, and freeze-drying to obtain guanidinoacetic acid;
(3) oxidation of guanidino glycogen: weighing 1g of guanidino glycogen, dissolving the guanidino glycogen in 50ml of deionized water, adding 297.2mg of sodium periodate after complete dissolution, reacting for 1 hour in a dark place, adding 790 mu L of ethylene glycol to stop the reaction when the reaction is finished, dialyzing the product in a 3500 dialysis bag for 48 hours, and freeze-drying to obtain the guanidino glycogen oxide.
The prepared guanidino glycogen oxide is characterized, glycogen, aminated glycogen, guanidino glycogen and guanidino glycogen oxide samples are respectively dissolved in heavy water (D2O), a 1H-NMR spectrum of the glycogen, aminated glycogen, guanidino glycogen and guanidino glycogen oxide samples is detected by a nuclear magnetic resonance spectrometer, and the result is shown in figure 3, and the two hydrogen atoms (2.9ppm and 3.2ppm) on methylene of ethylenediamine appear in a nuclear magnetic diagram of new aminated glycogen after the ethylenediamine is grafted; after guanidinoacetic acid conjugation, the original two methylene peaks in the product became 3.22ppm for one and 3.46ppm for the other due to changes in the chemical environment around the methylene group. A methylene peak is increased by 3.54ppm due to introduction of guanidino; aldehyde group is formed in the last ring-opening oxidation, and characteristic peak is present at 8.5-8.8 ppm.
Measuring and calculating the content of amino in the aminated glycogen by using an electric conductance titration method, and calculating to obtain the aminated glycogen with the amino content of 2.65 percent
The content of guanidino in guanidino glycogen is determined and calculated by adopting sakakou reaction, and the content of guanidino in guanidino glycogen is 7.20 mu g/mg by calculation.
And (3) measuring and calculating the content of aldehyde groups in the guanidino glycogen by a titration method, wherein the content of aldehyde groups in the guanidino glycogen is 7.47 percent through calculation.
The charging condition of each particle (1mg/mL) is measured by a potentiometer, and the results of measuring the potentials of the glycogen, the aminated glycogen, the guanidino glycogen and the guanidino glycogen oxide particles are shown in a figure 4, so that the glycogen, the guanidino glycogen and the guanidino glycogen oxide are originally negatively charged, and the aminated glycogen, the guanidino glycogen and the guanidino glycogen oxide are modified to have positive charges and can generate electrostatic interaction with hydroxyapatite with negative charges.
And observing the apparent morphological characteristics and the particle size of the guanidino glycogen oxide particles by using a transmission electron microscope, and determining the particle size and the distribution of the guanidino glycogen oxide particles by using a particle size analyzer, wherein the particle size is about 120nm, and the distribution is concentrated.
Example 2
The preparation of natural polymer hydrogel based on collagen comprises the following steps:
1) preparation of guanidino-oxidized glycogen: the same as example 1;
2) preparation of collagen solution: adding 11.25mg of collagen into every 1mL of water, and uniformly stirring at 37 ℃ to obtain a collagen solution;
3) preparation of guanidino glycogen oxide solution doped with nano-hydroxyapatite: adding 37.5mg of guanidino glycogen into 1mL of water, uniformly stirring to obtain a guanidino glycogen oxide solution, and adding 0mg of nano-hydroxyapatite to obtain a guanidino glycogen oxide solution doped with nano-hydroxyapatite (0 mg);
4) preparation of hydrogel: according to the volume, 2 parts of collagen solution and 1 part of guanidino glycogen oxide solution doped with nano-hydroxyapatite are taken, the guanidino glycogen oxide solution doped with the nano-hydroxyapatite is poured into the collagen solution under electric stirring (300rpm, the same below), the collagen solution and the guanidino glycogen oxide solution are uniformly mixed at 37 ℃, and the mixture is poured into a mold. And adding a carbonate buffer solution dropwise on the surface of the mixed solution, standing for 12h, taking out from a mold, reducing for 2h by using 4mL of 0.05 wt% sodium borohydride solution, washing with water for 3 times, and irradiating under an ultraviolet lamp for 2h for sterilization to obtain the natural polymer hydrogel based on collagen.
Example 3
Preparation of natural polymer hydrogel based on collagen and induction of MSCs differentiation, comprising the following steps:
1) preparation of guanidino-oxidized glycogen: the same as example 1;
2) preparation of collagen solution: adding 15mg of collagen into every 1mL of water, and uniformly stirring at 37 ℃ to obtain a collagen solution;
3) preparation of guanidino glycogen oxide solution doped with nano-hydroxyapatite: adding 50mg of guanidino glycogen into 1mL of water, uniformly stirring to obtain a guanidino glycogen oxide solution, and adding 0mg of nano-hydroxyapatite to obtain a guanidino glycogen oxide solution doped with nano-hydroxyapatite (0 mg);
4) the preparation of the hydrogel comprises the steps of taking 2 parts of collagen solution and 1 part of guanidino glycogen oxide solution doped with nano-hydroxyapatite by volume, pouring the guanidino glycogen oxide solution doped with the nano-hydroxyapatite into the collagen solution under electric stirring, uniformly mixing at 37 ℃, and pouring into a mold. And adding a carbonate buffer solution dropwise on the surface of the mixed solution, standing for 12h, taking out from a mold, reducing for 2h by using 4mL of 0.05 wt% sodium borohydride solution, washing with water for 3 times, and irradiating under an ultraviolet lamp for 2h for sterilization to obtain the natural polymer hydrogel based on collagen.
Example 4
The preparation of natural polymer hydrogel based on collagen comprises the following steps:
1) preparation of guanidino-oxidized glycogen: the same as example 1;
2) preparation of collagen solution: adding 18.75mg of collagen into every 1mL of water, and uniformly stirring at 37 ℃ to obtain a collagen solution;
3) preparation of guanidino glycogen oxide solution doped with nano-hydroxyapatite: adding 62.5mg of guanidino glycogen oxide into every 1mL of water, uniformly stirring to obtain a guanidino glycogen oxide solution, and adding 0mg of nano-hydroxyapatite into the guanidino glycogen oxide solution to obtain a guanidino glycogen oxide solution doped with nano-hydroxyapatite (0 mg);
4) the preparation of the hydrogel comprises the steps of taking 2 parts of collagen solution and 1 part of guanidino glycogen oxide solution doped with nano-hydroxyapatite by volume, pouring the guanidino glycogen oxide solution doped with the nano-hydroxyapatite into the collagen solution under electric stirring, uniformly mixing at 37 ℃, and pouring into a mold. And adding a carbonate buffer solution dropwise on the surface of the mixed solution, standing for 12h, taking out, reducing for 2h by using 4mL of 0.05 wt% sodium borohydride solution, washing for 3 times, and irradiating under an ultraviolet lamp for 2h for sterilization to obtain the natural polymer hydrogel based on collagen.
Example 5
The preparation of natural polymer hydrogel based on collagen comprises the following steps:
1) preparation of guanidino-oxidized glycogen: the same as example 1;
2) preparation of collagen solution: adding 22.5mg of collagen into every 1mL of water, and uniformly stirring at 37 ℃ to obtain a collagen solution;
3) preparation of guanidino glycogen oxide solution doped with nano-hydroxyapatite: adding 75mg of guanidino glycogen oxide into every 1mL of water, uniformly stirring to obtain a guanidino glycogen oxide solution, and adding 0mg of nano hydroxyapatite into the guanidino glycogen oxide solution to obtain a guanidino glycogen oxide solution doped with nano hydroxyapatite (0 mg);
4) preparation of hydrogel: according to the volume, 2 parts of collagen solution and 1 part of guanidino glycogen oxide solution doped with nano-hydroxyapatite are taken, the guanidino glycogen oxide solution doped with the nano-hydroxyapatite is poured into the collagen solution under electric stirring, the collagen solution and the guanidino glycogen oxide solution are uniformly mixed at 37 ℃, and the mixture is poured into a mold. And adding a carbonate buffer solution dropwise on the surface of the mixed solution, standing for 12h, taking out from a mold, reducing for 2h by using 4mL of 0.05 wt% sodium borohydride solution, washing with water for 3 times, and irradiating under an ultraviolet lamp for 2h for sterilization to obtain the natural polymer hydrogel based on collagen.
Example 6
Preparation of natural polymer hydrogel based on collagen and induction of MSCs differentiation, comprising the following steps:
1) preparation of guanidino-oxidized glycogen: the same as example 1;
2) preparation of collagen solution: adding 11.25mg of collagen into every 1mL of water, and uniformly stirring at 37 ℃ to obtain a collagen solution;
3) preparation of guanidino glycogen oxide solution doped with nano-hydroxyapatite: adding 50mg of guanidino glycogen into every 1mL of water, uniformly stirring to obtain a guanidino glycogen oxide solution, and adding 0mg of nano hydroxyapatite into the guanidino glycogen oxide solution to obtain a guanidino glycogen oxide solution doped with nano hydroxyapatite;
4) preparation of hydrogel: according to the volume, 2 parts of collagen solution and 1 part of guanidino glycogen oxide solution doped with nano-hydroxyapatite are taken, the guanidino glycogen oxide solution doped with the nano-hydroxyapatite is poured into the collagen solution under electric stirring, the collagen solution and the guanidino glycogen oxide solution are uniformly mixed at 37 ℃, and the mixture is poured into a mold. And adding a carbonate buffer solution dropwise on the surface of the mixed solution, standing for 12h, taking out from a mold, reducing for 2h by using 4mL of 0.05 wt% sodium borohydride solution, washing with water for 3 times, and irradiating under an ultraviolet lamp for 2h for sterilization to obtain the natural polymer hydrogel based on collagen.
Example 7
Preparation of natural polymer hydrogel based on collagen and induction of MSCs differentiation, comprising the following steps:
1) preparation of guanidino-oxidized glycogen: the same as example 1;
2) preparation of collagen solution: adding 18.75mg of collagen into every 1mL of water, and uniformly stirring at 37 ℃ to obtain a collagen solution;
3) preparation of guanidino glycogen oxide solution doped with nano-hydroxyapatite: adding 50mg of guanidino glycogen into every 1mL of water, uniformly stirring to obtain a guanidino glycogen oxide solution, and adding 0mg of nano hydroxyapatite into the guanidino glycogen oxide solution to obtain a guanidino glycogen oxide solution doped with nano hydroxyapatite;
4) preparation of hydrogel: according to the volume, 2 parts of collagen solution and 1 part of guanidino glycogen oxide solution doped with nano-hydroxyapatite are taken, the guanidino glycogen oxide solution doped with the nano-hydroxyapatite is poured into the collagen solution under electric stirring, the collagen solution and the guanidino glycogen oxide solution are uniformly mixed at 37 ℃, and the mixture is poured into a mold. Adding carbonate buffer solution to cover for 12h, taking out, reducing with 4mL of 0.05 wt% sodium borohydride solution for 2h, washing with water for 3 times, and sterilizing under ultraviolet lamp for 2h to obtain the natural polymer hydrogel based on collagen.
Example 8
Preparation of natural polymer hydrogel based on collagen and induction of MSCs differentiation, comprising the following steps:
1) preparation of guanidino-oxidized glycogen: the same as example 1;
2) preparation of collagen solution: adding 22.5mg of collagen into every 1mL of water, and uniformly stirring at 37 ℃ to obtain a collagen solution;
3) preparation of guanidino glycogen oxide solution doped with nano-hydroxyapatite: adding 50mg of guanidino glycogen into every 1mL of water, uniformly stirring to obtain a guanidino glycogen oxide solution, and adding 0mg of nano hydroxyapatite into the guanidino glycogen oxide solution to obtain a guanidino glycogen oxide solution doped with nano hydroxyapatite;
4) preparation of hydrogel: according to the volume, 2 parts of collagen solution and 1 part of guanidino glycogen oxide solution doped with nano-hydroxyapatite are taken, the guanidino glycogen oxide solution doped with the nano-hydroxyapatite is poured into the collagen solution under electric stirring, the collagen solution and the guanidino glycogen oxide solution are uniformly mixed at 37 ℃, and the mixture is poured into a mold. And adding a carbonate buffer solution dropwise on the surface of the mixed solution, standing for 12h, taking out from a mold, reducing for 2h by using 4mL of 0.05 wt% sodium borohydride solution, washing with water for 3 times, and irradiating under an ultraviolet lamp for 2h for sterilization to obtain the natural polymer hydrogel based on collagen.
Example 9
Preparation of natural polymer hydrogel based on collagen and induction of MSCs differentiation, comprising the following steps:
1) preparation of guanidino-oxidized glycogen: the same as example 1;
2) preparation of collagen solution: adding 15mg of collagen into every 1mL of water, and uniformly stirring at 37 ℃ to obtain a collagen solution;
3) preparation of guanidino glycogen oxide solution doped with nano-hydroxyapatite: adding 50mg of guanidino glycogen into 1mL of water, uniformly stirring to obtain a guanidino glycogen oxide solution, and adding 5mg of nano hydroxyapatite into the guanidino glycogen oxide solution to obtain a guanidino glycogen oxide solution doped with nano hydroxyapatite;
4) preparation of hydrogel: according to the volume, 2 parts of collagen solution and 1 part of guanidino glycogen oxide solution doped with nano-hydroxyapatite are taken, the guanidino glycogen oxide solution doped with the nano-hydroxyapatite is poured into the collagen solution under electric stirring, the collagen solution and the guanidino glycogen oxide solution are uniformly mixed at 37 ℃, and the mixture is poured into a mold. And adding a carbonate buffer solution dropwise on the surface of the mixed solution, standing for 12h, taking out from a mold, reducing for 2h by using 4mL of 0.05 wt% sodium borohydride solution, washing with water for 3 times, and irradiating under an ultraviolet lamp for 2h for sterilization to obtain the natural polymer hydrogel based on collagen.
Example 10
Preparation of natural polymer hydrogel based on collagen and induction of MSCs differentiation, comprising the following steps:
1) preparation of guanidino-oxidized glycogen: the same as example 1;
2) preparation of collagen solution: adding 15mg of collagen into every 1mL of water, and uniformly stirring at 37 ℃ to obtain a collagen solution;
3) preparation of guanidino glycogen oxide solution doped with nano-hydroxyapatite: adding 50mg of guanidino glycogen into 1mL of water, uniformly stirring to obtain a guanidino glycogen oxide solution, and adding 10mg of nano hydroxyapatite into the guanidino glycogen oxide solution to obtain a guanidino glycogen oxide solution doped with nano hydroxyapatite;
4) preparation of hydrogel: according to the volume, 2 parts of collagen solution and 1 part of guanidino glycogen oxide solution doped with nano-hydroxyapatite are taken, the guanidino glycogen oxide solution doped with the nano-hydroxyapatite is poured into the collagen solution under electric stirring, the collagen solution and the guanidino glycogen oxide solution are uniformly mixed at 37 ℃, and the mixture is poured into a mold. And adding a carbonate buffer solution dropwise on the surface of the mixed solution, standing for 12h, taking out from a mold, reducing for 2h by using 4mL of 0.05 wt% sodium borohydride solution, washing with water for 3 times, and irradiating under an ultraviolet lamp for 2h for sterilization to obtain the natural polymer hydrogel based on collagen.
Example 11
Preparation of natural polymer hydrogel based on collagen and induction of MSCs differentiation, comprising the following steps:
1) preparation of guanidino-oxidized glycogen: the same as example 1;
2) preparation of collagen solution: adding 15mg of collagen into every 1mL of water, and uniformly stirring at 37 ℃ to obtain a collagen solution;
3) preparation of guanidino glycogen oxide solution doped with nano-hydroxyapatite: adding 50mg of guanidino glycogen into 1mL of water, uniformly stirring to obtain a guanidino glycogen oxide solution, and adding 25mg of nano hydroxyapatite into the guanidino glycogen oxide solution to obtain a guanidino glycogen oxide solution doped with nano hydroxyapatite;
4) preparation of hydrogel: according to the volume, 2 parts of collagen solution and 1 part of guanidino glycogen oxide solution doped with nano-hydroxyapatite are taken, the guanidino glycogen oxide solution doped with the nano-hydroxyapatite is poured into the collagen solution under electric stirring, the collagen solution and the guanidino glycogen oxide solution are uniformly mixed at 37 ℃, and the mixture is poured into a mold. And adding a carbonate buffer solution dropwise on the surface of the mixed solution, standing for 12h, taking out from a mold, reducing for 2h by using 4mL of 0.05 wt% sodium borohydride solution, washing with water for 3 times, and irradiating under an ultraviolet lamp for 2h for sterilization to obtain the natural polymer hydrogel based on collagen.
After guanidino glycogen oxide and nano-hydroxyapatite are mixed in different ratios (10:1 and 1:10), the obtained guanidino glycogen oxide solution doped with nano-hydroxyapatite is subjected to particle size measurement, and the result is shown in fig. 5 (the ratios are mass ratios), so that the guanidino glycogen oxide solution with higher concentration can enable the nano-hydroxyapatite to be better aggregated. (the particle size of guanidino-oxide glycogen is slightly larger than its true size due to the surrounding aggregated hydrated layer at this time because of the lower concentration.)
After guanidino-oxidized glycogen and nano-hydroxyapatite were mixed at a mass ratio of 10:1 (the solution obtained in step (3) in example 9, a sample was prepared by appropriate dilution), and the structure thereof was observed by a transmission electron microscope, and as a result, as shown in fig. 6, it was found that aggregation of hydroxyapatite did occur around guanidino-oxidized glycogen.
Compression experiment: to evaluate the mechanical strength of the hydrogels, cylindrical gel samples of 10mm diameter and about 8mm thickness were prepared and subjected to uniaxial compressive stress-strain tests on a universal mechanical tester with a 50N weighing cell at a crosshead speed of 0.2 mm/min. Stress is represented by sigma ═ Load/pi r2And (d) determining, wherein r is the radius of the sample before compression. The strain epsilon is expressed as a change in height relative to the initial height of the sample. The final stress value was calculated as the amount of deformation (non-fractured sample) to fracture or each compression to 80% and the corresponding compression modulus (compression modulus is the slope of the linear interval during compression) was calculated.
As shown in FIG. 7, the compressive modulus of the hydrogel of natural polymer based on collagen obtained in examples 2 to 11 of the present invention is less than 80kPa, and is located in the region (about 80kPa) where the matrix hardness promotes cartilage differentiation, as seen from the compressive modulus of the hydrogel obtained in examples 2, 3, 6 and 7. On the other hand, the hydrogels prepared in examples 5, 10 and 11 had a hardness of more than 190kPa in the region (about 190kPa) where the matrix hardness promoted osteogenic differentiation. The compression modulus of the hydrogels prepared in the remaining examples is located between the interval of matrix stiffness promoting cartilage differentiation and osteogenic differentiation.
The hydrogel prepared in example 9 was subjected to a cyclic compression experiment, and the amount of deformation during each compression was 50%, and the structure is shown in fig. 8. It can be seen that the gel retains its previous strength after multiple compression experiments on the gel. The gel has certain resistance to external pressure and can be maintained for a long service time.
Cytotoxicity experiments: the cytotoxicity of the collagen-based natural polymer hydrogels of examples 2 to 11 on 3T3 fibroblasts was measured by MTT method. The specific operation is as follows: freeze-drying each gel, performing ultraviolet sterilization on the freeze-dried gel for 0.5h, and soaking the gel in a cell culture medium at a concentration of 5mg/mL, wherein the leaching liquor is used for culturing cells; spreading 3T3 cells with good growth state in 96-well plate at 3000/well at 37 deg.C and 5% CO2Culturing in an incubator for 24 hours; after the cells were attached to the wall, 100 μ l of the leaching solution of each different gel was added, 6 gels were set in parallel and a set of blank controls was set for each well plate. After the cells were cultured for 24/72/108 hours, the medium was discarded, 100. mu.l of 0.5mg/ml MTT was added, and the mixture was placed in a cell incubator and cultured for 4 hours to terminate the culture. The supernatant in the well plate was aspirated, 100. mu.l of dimethyl sulfoxide was added to each well, the mixture was allowed to stand for 15 minutes, and the absorbance at 570nm was measured with a microplate reader.
Cytotoxicity (%) ═ absorbance of cells in gel group/absorbance of cells in blank group 100%.
The experimental results are as follows: the cytotoxicity of the collagen-based natural polymer hydrogel of the present invention against 3T3 cells is shown in fig. 9. Therefore, the collagen-based natural polymer hydrogel has the effect of promoting the proliferation of 3T3 fibroblasts, and the higher the collagen content is, the better the proliferation effect is. In addition, the addition of the nano-hydroxyapatite can also promote proliferation.
MSCs differentiation assay: the content of ALP in Mesenchymal Stem Cells (MSCs) grown on the collagen-based natural polymer hydrogels prepared in examples 3, 4, 5, 7, 8, 9, 10, and 11 was measured by an alkaline phosphatase (ALP) content quantitative assay, and was used to indicate the differentiation of the MSCs into osteoblasts. Specifically, the hydrogel is laid in a 24-pore plate, 3 gels are arranged in parallel, and after ultraviolet sterilization is carried out for 0.5 hour, the gel is soaked in phosphate buffer solution for 1 hour; collecting MSCs with good growth state, and spreading at density of 50000/holeOn hydrogel in 24-well plates, 37 ℃, 5% CO2After 7/14 days of culture in the incubator, the medium was discarded, the cells were lysed by adding 1% triton solution for half an hour after washing twice with phosphate buffer, and then the ALP content was measured with an ALP quantitative determination kit, and the absorbance at 405nm was measured with a microplate reader as a unit, 3 times per well in parallel. Meanwhile, the total protein content in the solution is determined by using a BCA kit, the absorption value of the solution at 562nm is detected by using a microplate reader, and a standard curve (R) made by a protein standard substance is passed2>0.999) to calculate the total protein content:
ALP content as OD405Total protein content.
The experimental results are as follows: the content of alkaline phosphatase (ALP) in the mesenchymal stem cells grown on the collagen-based natural polymer hydrogel of the present invention is shown in fig. 10. Therefore, the collagen-based natural polymer hydrogel plays a role in promoting the differentiation of MSCs into osteoblasts, and the higher the compression modulus is, the more obvious the differentiation effect is. In addition, the increase of the nano hydroxyapatite can promote osteogenic differentiation.
Comparative example 1
The artificial bone material prepared by directly mixing collagen and nano hydroxyapatite by the traditional method has the following defects: the state is single, most of the materials are solid, the biocompatibility is poor, and the filling of the bone defect part is difficult; the collagen is degraded quickly when existing in vivo for a short time, and the hydroxyapatite is settled and is not beneficial to the adhesion and growth of cells on the hydroxyapatite.
Comparative example 2
In example 1, the carbodiimide hydrochloride and the N-hydroxysuccinimide in step (2) need more than 1 time of the substance amount of the guanidinoacetic acid, and if the carbodiimide hydrochloride and the N-hydroxysuccinimide in the process are less than 1 time of the substance amount of the guanidinoacetic acid, the grafting ratio of the guanidino in the final result is almost 0, and the positive charge of the guanidino glycogen is hardly increased, which is not beneficial to the subsequent and nano-hydroxyapatite effect.
Comparative example 3
Hydrogel materials were prepared when conventional glycogen was used in place of the guanidyl oxide glycogen of the present invention, in the same manner as in example 9 of the present invention;
the preparation of natural polymer hydrogel based on collagen comprises the following steps:
1) preparation of collagen solution: adding 15mg of collagen into every 1mL of water, and uniformly stirring at 37 ℃ to obtain a collagen solution;
2)3) preparation of glycogen solution doped with nano-hydroxyapatite: adding 50mg of glycogen into every 1mL of water, uniformly stirring to obtain a glycogen solution, and then adding 5mg of nano-hydroxyapatite into the glycogen solution to obtain a glycogen solution doped with the nano-hydroxyapatite;
4) preparation of hydrogel: according to the volume, 2 parts of collagen solution and 1 part of glycogen solution doped with nano-hydroxyapatite are taken, the glycogen solution doped with the nano-hydroxyapatite is poured into the collagen solution under electric stirring, mixed evenly at 37 ℃, and poured into a mould. And adding a carbonate buffer solution dropwise on the surface of the mixed solution, standing for 12h, taking out from the mold, reducing with 0.05% sodium borohydride solution for 2h, washing with water for 3 times, and irradiating under an ultraviolet lamp for 2h for sterilization to obtain the collagen-based natural polymer hydrogel.
The hydrogel is not chemically crosslinked, and although the hydrogel is in a gel state at room temperature, the hydrogel cannot maintain the gel state at 37 ℃, becomes a viscous liquid and cannot support subsequent experiments.
The hydrogel material prepared has the advantages that since unmodified glycogen has negative charges and cannot generate electrostatic attraction on nano-hydroxyapatite with the same negative charges, ordered cross-linking cannot be formed inside the hydrogel material, and organic components and inorganic components are still separated. Can not exist stably in the body, the collagen can be degraded quickly, and the hydroxyapatite can be settled, which is not beneficial to the adhesion and growth of cells on the hydroxyapatite.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A guanidino glycogen oxide characterized by the structural formula:
wherein m is more than or equal to 1, n is more than or equal to 1, i is more than or equal to 1, and the average molecular weight is 8000-1000 kDa.
2. The method for producing guanidino-oxide glycogen according to claim 1, characterized by comprising the steps of:
(1) electropositivity of glycogen: dissolving glycogen in dimethyl sulfoxide to form a solution with the mass-to-volume ratio of 0.5-2%, then adding N, N-carbonyl diimidazole with the amount of 1-20 times that of the glycogen, stirring for 0.5-4 hours under the protection of inert atmosphere, then dissolving ethylenediamine with the amount of 5-50 times that of the glycogen in dimethyl sulfoxide to obtain an ethylenediamine solution, dropwise adding the ethylenediamine solution into the activated reaction solution, reacting for 12-24 hours under the protection of inert atmosphere, and purifying and drying to obtain the aminated glycogen;
(2) grafting a guanidino group on aminated glycogen: dissolving guanidinoacetic acid in water to obtain a guanidinoacetic acid solution with the mass-to-volume ratio of 0.5-2% and adjusting the pH to 5-6, adding carbodiimide hydrochloride in an amount which is 1-5 times of the amount of the guanidinoacetic acid and N-hydroxysuccinimide in an amount which is 1-5 times of the amount of the guanidinoacetic acid into the guanidinoacetic acid solution, adding aminated glycogen prepared in the step (1) in an amount which is 0.1-1 times of the amount of the guanidinoacetic acid after activating for 0.5-4 hours, adjusting the pH of the solution to 7-8, reacting for 12-16 hours, and purifying and drying to obtain guanidino glycogen;
(3) oxidation of guanidino glycogen: and (3) dissolving a proper amount of the guanidino glycogen prepared in the step (2) in water to obtain a guanidino glycogen solution with the mass-volume ratio of 0.5-2%, adding sodium periodate with the amount of 1-5 times that of the guanidino glycogen, reacting for 1-3 hours in a dark place, purifying, and drying to obtain the guanidino glycogen oxide.
3. The production method according to claim 2, wherein in the step (3), the reaction is terminated by adding ethylene glycol in an amount of 2 to 10 times that of the substance of guanidino glycogen at the end of the reaction.
4. A method for preparing a collagen-based hydrogel, comprising: respectively preparing an aqueous collagen solution with the concentration of 11.25-22.5mg/mL and the guanidino glycogen oxide solution of claim 1 with the concentration of 37.5-75mg/mL, and then adding 0-25mg of nano-hydroxyapatite into each 1mL of the guanidino glycogen oxide solution to obtain a guanidino glycogen oxide solution doped with nano-hydroxyapatite; mixing a collagen water solution and a guanidino glycogen oxide solution doped with nano-hydroxyapatite according to a volume ratio of 2:1, dropwise adding a carbonate buffer solution on the mixed solution after 15-60 minutes, standing for 2-24 hours, reducing for 0.5-2 hours by using a sodium borohydride solution with a mass volume fraction of 0.05-5%, washing with water, and sterilizing to obtain the collagen-based natural polymer hydrogel.
5. The method of claim 4, wherein the carbonate buffer is sodium carbonate-sodium bicarbonate solution with a concentration of 0.5 mol/L.
6. The method for preparing a collagen-based hydrogel according to claim 4 or 5, wherein said collagen is provided by Wunny Bedy bioengineering GmbH.
7. A collagen-based natural polymer hydrogel produced by the method for producing a collagen-based hydrogel according to any one of claims 4 to 6.
8. Nutraceutical, pharmaceutical comprising a collagen-based hydrogel according to claim 7.
9. An artificial bone comprising the collagen-based hydrogel of claim 7.
10. Use of the collagen-based hydrogel of claim 7 for the differentiation of mesenchymal stem cells.
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Denomination of invention: A collagen based natural polymer hydrogel and its preparation method Granted publication date: 20210625 Pledgee: Wuxi rural commercial bank Limited by Share Ltd. Pledgor: WUXI BIOT BIO-TECHNOLOGY CO.,LTD. Registration number: Y2024980017171 |