CN108003360B - Preparation method of II type collagen hydrogel for inducing stem cell chondrogenic differentiation - Google Patents

Preparation method of II type collagen hydrogel for inducing stem cell chondrogenic differentiation Download PDF

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CN108003360B
CN108003360B CN201711125169.XA CN201711125169A CN108003360B CN 108003360 B CN108003360 B CN 108003360B CN 201711125169 A CN201711125169 A CN 201711125169A CN 108003360 B CN108003360 B CN 108003360B
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孙静
杨棵
卫丹
范红松
张兴栋
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Abstract

The invention discloses a preparation method of II type collagen hydrogel for inducing stem cell chondrogenic differentiation, which comprises the steps of firstly synthesizing photocrosslinkable II type collagen with photocrosslinkability and keeping the triple helix conformation integrity of the II type collagen through amidation reaction of lysine-amino of a side chain of the II type collagen and methacrylic anhydride; and then under the action of a photoinitiator, initiating the polymerization and crosslinking of the photo-crosslinkable II type collagen to form the adjustable single-component II type collagen hydrogel with certain mechanical strength, thereby providing a bionical differentiation microenvironment for stem cells.

Description

Preparation method of II type collagen hydrogel for inducing stem cell chondrogenic differentiation
Technical Field
The invention belongs to the technical field of biomedical materials, and relates to a preparation method and application of an inductive hydrogel material for cartilage defect repair.
Background
Given the avascular and low overall metabolic rate and active cell density of articular cartilage tissue, patients have a very limited ability to self-repair when they suffer from cartilage related diseases or trauma. Common clinical means for cartilage repair comprise bone marrow stimulation, osteochondral transplantation, prosthesis replacement and the like, but the means usually cause the formation of fibrocartilage tissues, and the structural composition and mechanical strength of fibrocartilage tissues are greatly different from those of natural hyaline cartilage tissues, so that the common clinical means for cartilage repair are difficult to realize long-term effective treatment effect. In the cell therapy based on tissue engineering, the cartilage micro-environment is favorably reconstructed by inoculating the cartilage cells or cartilage precursor cells to the bionics compatible bracket; wherein the biomimetic scaffold is crucial for chondrocyte phenotype maintenance and Bone marrow Mesenchymal stem cells (BMSCs) cartilage differentiation.
Type II collagen is an important component of the chondrocyte extracellular matrix, and has excellent biocompatibility and biodegradability. However, type II collagen has a poor fiber assembly ability and is difficult to gel, so that it cannot stably provide cells with a necessary microenvironment for proliferation and differentiation for a long period of time. The common crosslinking mode for preparing the gel by taking the type II collagen as the raw material is mainly carried out by glutaraldehyde and genipin, EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride)/NHS (N-hydroxysuccinimide) and the like are covalently cross-linked with lyophilized collagen II sponge to obtain a scaffold material having a certain mechanical strength (C. -S.Ko, J. -P.Huang, C. -W.Huang and I. -M.Chu, Journal of bioscience and bioengineering,2009,107,177-182, X.ZHou, Y.Tao, J.Wang, D.Liu, C.Liang, H.Li and Q.Chen, Journal of biological Materials Research Part A,2016, 1687-16993, J.Pieper, P.Van der Kraan, T.Hafmans, J.Kamp, P.Buma, J.Van. san W.W.12n, K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.. However, the stent material prepared by the method has potential cytotoxicity due to the fact that glutaraldehyde, genipin and EDC/NHS are toxic; and the sponge structure is lack of bionics to the three-dimensional cartilage microenvironment, and can not provide a proper growth and differentiation environment for cells. Furthermore, the use of composite gel materials is not sufficient to highlight the important role of type II collagen in influencing and directing the cell behavior.
Disclosure of Invention
The invention aims to provide a preparation method of type II collagen hydrogel for inducing stem cells to chondrogenic differentiation, aiming at overcoming the defects in the prior art, so as to obtain biocompatible hydrogel based on single components of type II collagen, construct a three-dimensional biological bionic microenvironment to better simulate cartilage extracellular matrix, and further realize the purpose of inducing BMSCs to chondrogenic differentiation.
The preparation method of the II type collagen hydrogel for inducing the chondrogenic differentiation of stem cells, provided by the invention, comprises the steps of taking II type collagen extracted from natural cartilage as a cartilage induction matrix, and synthesizing photocrosslinkable II type collagen with photocrosslinkability and maintaining the triple helix conformation integrity of the II type collagen through amidation reaction of amino on lysine of a side chain of the II type collagen and methacrylic anhydride; and then initiating the polymerization crosslinking of the photo-crosslinkable II type collagen to form the adjustable single-component II type collagen hydrogel with certain mechanical strength under the catalysis of the photoinitiator, which comprises the following specific steps:
(1) preparing type II collagen solution
Dissolving type II collagen in acid solution with pH value of 1-3 at 0-4 deg.C under normal pressure to obtain type II collagen solution with mass concentration of 1-4 mg/mL;
(2) synthesis of photo-crosslinkable type II collagen
Regulating the pH value of the II type collagen solution to be more than 7 and less than or equal to 8 by using a phosphate buffer solution or/and a sodium hydroxide solution at normal pressure and 0-4 ℃, then adding methacrylic anhydride, stirring and reacting for 2-12h at normal pressure and 0-4 ℃, supplementing a sodium hydroxide solution in the reaction process to ensure that the pH value of a reaction system is more than 7 and less than or equal to 8, and metering the adding amount of the methacrylic anhydride according to the molar ratio of amino in the II type collagen solution to the methacrylic anhydride of 1 (0.2-5);
removing unreacted methacrylic anhydride from the obtained reaction liquid after the reaction is finished, then freezing and drying the reaction liquid from which the unreacted methacrylic anhydride is removed to obtain the photocrosslinkable II-type collagen, or filling the reaction liquid from which the unreacted methacrylic anhydride is removed into a dialysis bag, and immersing the dialysis bag containing the reaction liquid into a polyethylene glycol aqueous solution with the mass concentration of 10-50% at the normal pressure and the temperature of 0-4 ℃ for concentrating for 4-24h to obtain a concentrated solution containing the photocrosslinkable II-type collagen;
(3) preparation of photo-crosslinked type II collagen hydrogel
Dissolving photo-crosslinkable II type collagen by using an acid solution with the pH value of 1-3 at the normal pressure and the temperature of 0-4 ℃ to obtain a photo-crosslinkable II type collagen solution, adjusting the pH value to 7.0-7.5 by using a sodium hydroxide solution, adding a photoinitiator into the photo-crosslinkable II type collagen solution after the pH value is adjusted to prepare a gel pre-polymerizing solution with the final concentration of the photo-crosslinkable II type collagen of 4-10mg/mL and the final concentration of the photoinitiator of 0.2-1mg/mL, and then placing the gel pre-polymerizing solution under the normal pressure and the room temperature for irradiating for 10-60s under ultraviolet light to obtain the II type collagen hydrogel;
or under the conditions of normal pressure and 0-4 ℃, regulating the pH value of the concentrated solution containing the photocrosslinkable II type collagen to 7.0-7.5 by using a sodium hydroxide solution, adding a photoinitiator into the photocrosslinkable II type collagen solution after the pH value is regulated to prepare a gel pre-polymerization solution with the photocrosslinkable II type collagen final concentration of 4-10mg/mL and the photoinitiator final concentration of 0.2-1mg/mL, and then placing the gel pre-polymerization solution under ultraviolet light at normal pressure and room temperature for irradiating for 10-60s to obtain the II type collagen hydrogel.
In the method for producing a collagen type II hydrogel for inducing stem cell chondrogenic differentiation, unreacted methacrylic anhydride in the obtained reaction solution is removed by dialysis after the reaction in the step (2) in the following manner: firstly, adding ethanol into an acid solution with the pH value of 1-3 to prepare a series of dialyzates, wherein the volume fraction of the ethanol in each dialyzate is sequentially reduced by 10 percent in the series of dialyzates, and the volume fraction of the ethanol in the dialyzate with the largest volume fraction of the ethanol is not more than 50 percent; then putting the dialysis bag containing the reaction solution into the dialysate to perform dialysis for a plurality of times at 0-4 ℃ under stirring, wherein the volume fraction of ethanol in the dialysate replaced by each dialysis is reduced by 10 percent, and the dialysis time is 3-7 days each time; finally, dialyzing the dialysis bag containing the reaction solution in an acid solution with the pH value of 2-3 for 1-3 days. The cut-off molecular weight of the dialysis bag is 8000-14000 daltons.
In the step (3), the photoinitiator is irgacure2959 (2-hydroxy-4 '- (2-hydroxyethoxy) -2-methylpropiophenone), irgacure184 (1-hydroxycyclohexyl phenyl ketone), irgacure127(1,1' - (methylenebis-4, 1-phenylene) bis [ 2-hydroxy-2-methyl-1-propanone ]), irgacure651 (benzoin dimethyl ether) or rose Bengal (tetrachlorotetraiodofluorescein disodium).
In the step (3), the amount of the acid solution is limited to at least completely dissolve the photocrosslinkable type II collagen. After the photoinitiator is added, a gel pre-polymerization solution meeting the requirements of the photo-linkable type II collagen concentration and the final concentration of the photoinitiator can be obtained by supplementing a phosphate buffer solution.
The preparation method of the II type collagen hydrogel for inducing the stem cell chondrogenic differentiation comprises the step (3) of controlling the ultraviolet light intensity to be 4-8W/cm2Under the condition of the light intensity, the polymerization crosslinking of the photocrosslinkable II collagen can be realized only by 10-60s of irradiation time, and the II type collagen hydrogel is obtained.
In the above method for preparing type II collagen hydrogel for inducing stem cell chondrogenic differentiation, in the steps (2) and (3), the molar concentration of the sodium hydroxide solution is 0.5-5M.
The preparation method of the II type collagen hydrogel for inducing the chondrogenic differentiation of the stem cells comprises the following steps of (2) and (3), wherein the molar concentration of the phosphate buffer solution is 0.2-2M; the phosphate buffer solution is disodium hydrogen phosphate solution or sodium dihydrogen phosphate solution.
In the method for preparing the collagen type II hydrogel for inducing the chondrogenic differentiation of the stem cells, the acid solution in the steps (1), (2) and (3) is a hydrochloric acid solution or an acetic acid solution.
When the II-type collagen hydrogel provided by the invention is applied to promoting osteogenic differentiation of stem cells, mesenchymal stem cells digested by trypsin and the prepolymerization solution obtained in the step (2) can be uniformly mixed, the obtained mixed solution is placed under ultraviolet light for irradiation to obtain the II-type collagen hydrogel wrapping the cells, and then the II-type collagen hydrogel wrapping the cells is cultured in a culture medium; when in use, the II type collagen hydrogel which is taken out from the culture medium and wraps the cells is implanted into corresponding tissues for culture so as to realize cartilage repair; or directly implanting the II type collagen hydrogel prepared in the step (3) into corresponding tissues for culture to realize cartilage repair.
Compared with the prior art, the invention has the following beneficial effects:
1. according to the method, through amidation reaction of amino on lysine on a side chain of the type II collagen and methacrylic anhydride, the type II collagen has photocrosslinking performance, the integrity of the original triple helix conformation of the type II collagen is kept, and the single-component type II collagen hydrogel with certain mechanical strength is obtained through further polymerization crosslinking, so that a bionic differentiation microenvironment is provided for stem cells.
2. The method of the invention utilizes the high reactivity of lysine-amino on the lysine of the side chain of the type II collagen to ensure that the type II collagen has the property of photocrosslinking, improves the defect of insufficient self-assembly capability of the type II collagen in a covalent crosslinking mode to be beneficial to the formation of later-stage hydrogel, and can realize the regulation and control of mechanical strength in a larger range by regulating the concentration of the gel pre-polymerization solution.
3. The II type collagen hydrogel prepared by the method has good cell compatibility because the II type collagen is from cartilage tissues, can promote chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells, and improves the expression of cartilage specific matrixes.
4. The preparation method of the II collagen hydrogel can be realized based on conventional equipment, and the used raw materials are nontoxic and environment-friendly, thereby being beneficial to realizing industrial production.
Drawings
FIG. 1 is the polyacrylamide gel electrophoresis detection analysis chart of natural type II collagen neutral solution.
FIG. 2 is a hydrogen nuclear magnetic resonance spectrum (1H-NMR) of the mixture, wherein (a) is a nuclear magnetic resonance spectrum of the natural type II collagen and the photocrosslinkable type II collagen prepared in example 1, (b) is a nuclear magnetic resonance spectrum of the photocrosslinkable type II collagen prepared in example 5, and (c) is a nuclear magnetic resonance spectrum of the photocrosslinkable type II collagen prepared in example 8Original hydrogen nuclear magnetic resonance spectrogram.
Fig. 3 is a graph of gelling effect and storage modulus, wherein (a) is a graph of gelling effect of a neutral solution of native type II collagen, (b) is a graph of gelling effect of the type II collagen hydrogel prepared in example 1, and (c) is a graph of storage modulus of the type II collagen hydrogel prepared in example 1, example 5 and example 8.
Fig. 4 is a graph showing the results of FDA/PI staining of cells cultured in collagen type II hydrogel encapsulated in rabbit bone marrow mesenchymal stem cells in application example 1, wherein (a) is a microscopic image of the cells after 1 day of culture, (b) is a microscopic image of the cells after 4 days of culture, (c) is a microscopic image of the cells after 7 days of culture, (d) is a microscopic image of the cells after 14 days of culture, and (e) is a microscopic image of the cells after 21 days of culture.
Fig. 5 is a microscope image of rabbit bone marrow mesenchymal stem cells packed in collagen type II hydrogel cultured in vivo for 14 days in application example 2, wherein (a) the microscope image is obtained by staining tissue sections with safranin o (so), and (b) the microscope image is obtained by staining tissue sections with Toluidine Blue (TB).
Detailed Description
The technical solutions of the present invention will be described in detail and fully with reference to the accompanying drawings, which are used for describing the embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the following examples, type II collagen, which is a kind of collagen, was purchased from Chondrex, USA, and subjected to NMR analysis, and the resultant NMR chart is shown in FIG. 2 (a). Firstly, the type II collagen is dissolved in a hydrochloric acid solution with the concentration of 20mM to prepare a type II collagen solution with the concentration of 2mg/mL, and then the pH value of the type II collagen solution is adjusted to 7 by using a disodium hydrogen phosphate buffer solution with the concentration of 0.2M and a sodium hydroxide solution with the concentration of 5M to obtain a natural type II collagen neutral solution, wherein the natural type II collagen neutral solution is in a solution state as shown in a figure 3 (a). Performing polyacrylamide gel electrophoresis detection on the obtained natural type II collagen neutral solution, wherein the detection result takes M (marker) and Col-I (type I collagen) as controls, the analysis result is shown in figure 1, the collagen molecular weight distribution of the type II collagen is characterized by three chains of alpha, beta and gamma, and the molecular weights of the beta and gamma chains are 250kDa and 300kDa respectively; compared with the heterogeneous type I collagen (two alpha bands exist), the alpha chain of the type II collagen belongs to homotrimer, so that only one alpha chain exists in the electrophoresis band.
In the examples described below, methacrylic anhydride, photoinitiator irgacure2959 was obtained from sigma corporation, usa and irgacure651 was obtained from BASF, germany.
Rabbit mesenchymal stem cells (BMSCs) can be extracted by conventional methods disclosed in the art [ see l.zhang, t.yuan, l.guo and x.zhang, Journal of biological Materials research patent a,2012,100,2717-2725 ], and the BMSCs used in the following examples are self-extracted by: injecting excessive pentobarbital sodium into newborn rabbit for killing, soaking in 0.5% (volume fraction) benzalkonium bromide for 10-20 min for sterilization, taking off tibia and femur under aseptic condition together with epiphyseal cartilage, removing muscle and fiber tissue, stripping cartilage tissue and exposing marrow cavity, extracting alpha-MEM culture medium containing 20% fetal calf serum with injector to flush marrow cavity (culture medium volume is 10 mL/dish), repeatedly blowing bone marrow tissue to make bone marrow cells fully dispersed in culture medium, supplementing 100U/mL penicillin and streptomycin (HyClone) with culture medium volume about 1%, placing at 37 deg.C and containing 5% (volume fraction) CO2Culturing for 24h in an adherent manner in the incubator, changing the culture solution to remove blood cells without adherence, adding the alpha-MEM culture medium containing 20% fetal calf serum again to continue culturing, changing the culture solution once every 2 days, and culturing primary BMSCs until 4 th day of passage. alpha-MEM medium was purchased from Hyclone, USA; the fetal bovine serum is Gibco fetal bovine serum from Life Technologies, USA.
Example 1
The steps of this example are as follows:
(1) preparing type II collagen solution
Type II collagen was dissolved in a 20mM hydrochloric acid solution at 4 ℃ under normal pressure for 12 hours while stirring the solution to prepare a type II collagen solution having a concentration of 2 mg/mL.
(2) Synthesis of photo-crosslinkable type II collagen
Adjusting the pH value of the II type collagen solution to 7.5 by using 0.2M disodium hydrogen phosphate buffer solution and 5M sodium hydroxide solution under the condition of normal pressure and 4 ℃, then adding methacrylic anhydride into the II type collagen solution after the pH value is adjusted according to the molar ratio of amino groups to methacrylic anhydride in collagen of 1:5, continuously stirring and reacting for 4 hours, and supplementing 5M sodium hydroxide solution in the reaction process to keep the pH value of the reaction system at 7.5.
After the reaction is finished, unreacted methacrylic anhydride in the obtained reaction liquid is removed through dialysis, and the specific mode is as follows: firstly, adding ethanol into a 20mM hydrochloric acid solution to prepare a dialysate with ethanol volume fractions of 10%, 20%, 30%, 40% and 50%; then putting the dialysis bag containing the obtained reaction liquid into the dialysate according to the sequence of the reduction of the volume fraction of the ethanol in the dialysate, and carrying out dialysis for a plurality of times under the condition of stirring at 4 ℃, wherein the dialysis time is 3-7 days each time; finally, the dialysis bag containing the resulting reaction solution was dialyzed in a 20mM hydrochloric acid solution for 2 days.
And (3) removing methacrylic anhydride, and freeze-drying the reaction solution to obtain the photo-crosslinkable II type collagen.
(3) Preparation of photo-crosslinked type II collagen hydrogel
Under the condition of normal pressure and 4 ℃, using 20mM hydrochloric acid solution to dissolve the photo-crosslinkable II collagen and prepare a photo-crosslinkable II collagen solution with the concentration of 10mg/mL, using 5M sodium hydroxide solution to adjust the pH value to 7.0, adding a photoinitiator irgacure2959 into the photo-crosslinkable II collagen solution after the pH value is adjusted to prepare a gel pre-polymerization solution with the final concentration of the photo-crosslinkable II collagen of 7mg/mL and the final concentration of the photoinitiator of 0.5mg/mL, and if the concentration of the gel pre-polymerization solution after the addition of the photoinitiator is higher, the requirements of the final concentrations of the photo-crosslinkable II collagen and the photoinitiator cannot be met, supplementing a corresponding volume of 0.2M disodium hydrogen phosphate buffer solution. The gel prepolymer was then exposed to an intensity of 8W/cm2Irradiating for 30 seconds under ultraviolet light to realize the curing of the pre-polymerized liquid and obtain the II type collagen hydrogel.
Example 2
The steps of this example are as follows:
(1) preparing type II collagen solution
Type II collagen was dissolved in a 20mM acetic acid solution at 0 ℃ under normal pressure for 12 hours while stirring to prepare a type II collagen solution having a concentration of 3 mg/mL.
(2) Synthesis of photo-crosslinkable type II collagen
Adjusting the pH value of the II type collagen solution to 8 by using 0.5M sodium dihydrogen phosphate buffer solution and 3M sodium hydroxide solution under the condition of normal pressure and 0 ℃, then adding methacrylic anhydride into the II type collagen solution after the pH value is adjusted according to the molar ratio of amino groups to methacrylic anhydride in collagen of 1:2.5, continuously stirring and reacting for 8 hours, and supplementing 3M sodium hydroxide solution in the reaction process to keep the pH value of a reaction system at 8.
After the reaction is finished, unreacted methacrylic anhydride in the obtained reaction liquid is removed through dialysis, and the specific mode is as follows: firstly, adding ethanol into a 20mM acetic acid solution to prepare a dialysate with ethanol volume fractions of 10%, 20%, 30%, 40% and 50%; then putting the dialysis bag containing the obtained reaction liquid into the dialysate according to the order of decreasing the volume fraction of ethanol in the dialysate, and dialyzing for 3-7 days each time under the condition of stirring at 0 ℃; finally, the dialysis bag containing the obtained reaction solution was dialyzed in a 20mM acetic acid solution for 1 day.
And (3) removing methacrylic anhydride, and freeze-drying the reaction solution to obtain the photo-crosslinkable II type collagen.
(3) Preparation of photo-crosslinked type II collagen hydrogel
Under the condition of normal pressure and 0 ℃, dissolving photo-crosslinkable II collagen by using 20mM acetic acid, preparing a photo-crosslinkable II collagen solution with the concentration of 10mg/mL, adjusting the pH value to 7.0 by using a 5M sodium hydroxide solution, adding a photoinitiator irgacure651 into the photo-crosslinkable II collagen solution after the pH value is adjusted, preparing a gel pre-polymerization solution with the final concentration of the photo-crosslinkable II collagen of 4mg/mL and the final concentration of the photoinitiator of 0.2mg/mL, and supplementing a corresponding volume of 0.5M sodium dihydrogen phosphate buffer solution if the concentration of the gel pre-polymerization solution after the addition of the photoinitiator is higher and cannot meet the requirements of the final concentrations of the photo-crosslinkable II collagen and the photoinitiator.The gel prepolymer was then exposed to a light intensity of 4W/cm2Irradiating for 60 seconds under ultraviolet light to realize the curing of the pre-polymerized liquid and obtain the II type collagen hydrogel.
Example 3
The steps of this example are as follows:
(1) preparing type II collagen solution
Type II collagen was dissolved in 100mM hydrochloric acid solution at 4 ℃ under normal pressure for 12 hours while stirring to prepare a type II collagen solution having a concentration of 4 mg/mL.
(2) Synthesis of photo-crosslinkable type II collagen
Adjusting the pH value of the II type collagen solution to 7.5 by using 1M disodium hydrogen phosphate buffer solution and 1M sodium hydroxide solution under the condition of normal pressure and 4 ℃, then adding methacrylic anhydride into the II type collagen solution after the pH value is adjusted according to the molar ratio of amino groups to methacrylic anhydride in collagen of 1:0.5, continuously stirring and reacting for 12 hours, and supplementing 1M sodium hydroxide solution in the reaction process to keep the pH value of the reaction system at 7.5.
After the reaction is finished, unreacted methacrylic anhydride in the obtained reaction liquid is removed through dialysis, and the specific mode is as follows: firstly, adding ethanol into 100mM hydrochloric acid solution to prepare dialysate with ethanol volume fractions of 10%, 20%, 30%, 40% and 50%; then putting the dialysis bag containing the obtained reaction liquid into the dialysate according to the sequence of the reduction of the volume fraction of the ethanol in the dialysate, and carrying out dialysis for a plurality of times under the condition of stirring at 4 ℃, wherein the dialysis time is 3-7 days each time; finally, the dialysis bag containing the resulting reaction solution was dialyzed against 100mM hydrochloric acid solution for 3 days.
And (3) immersing the dialysis bag containing the reaction solution after methacrylic anhydride removal into a PEG (polyethylene glycol) aqueous solution with the mass concentration of 10% for concentration for 24h at the temperature of 4 ℃ under normal pressure to obtain a concentrated solution containing the photocrosslinkable II type collagen.
(3) Preparation of photo-crosslinked type II collagen hydrogel
Adjusting pH of the concentrate containing photo-crosslinkable II collagen to 7.5 with 1M sodium hydroxide solution at 4 deg.C under normal pressure, adding photoinitiator irgacure2959 into the photo-crosslinkable II collagen solution after pH adjustment, and making into photo-crosslinkable II collagenThe final concentration of collagen is 10mg/mL, the final concentration of the photoinitiator is 1mg/mL, if the concentration of the gel pre-polymerization solution after the photoinitiator is added is higher, the requirements of photo-crosslinkable II type collagen and the final concentration of the photoinitiator cannot be met, and 1M disodium hydrogen phosphate buffer solution with the corresponding volume can be supplemented. The gel prepolymer was then exposed to an intensity of 8W/cm2Irradiating for 10 seconds under ultraviolet light to realize the curing of the pre-polymerized liquid and obtain the II type collagen hydrogel.
Example 4
The steps of this example are as follows:
(1) preparing type II collagen solution
Type II collagen was dissolved in a 10mM hydrochloric acid solution at 0 ℃ under normal pressure for 12 hours while stirring the solution to prepare a type II collagen solution having a concentration of 2 mg/mL.
(2) Synthesis of photo-crosslinkable type II collagen
Adjusting the pH value of the II type collagen solution to 7.5 by using 2M sodium dihydrogen phosphate buffer solution and 0.5M sodium hydroxide solution under the condition of normal pressure and 0 ℃, then adding methacrylic anhydride into the II type collagen solution after the pH value is adjusted according to the molar ratio of amino groups to methacrylic anhydride in collagen of 1:3, continuously stirring and reacting for 2 hours, and supplementing 0.5M sodium hydroxide solution in the reaction process to keep the pH value of the reaction system at 7.5.
After the reaction is finished, unreacted methacrylic anhydride in the obtained reaction liquid is removed through dialysis, and the specific mode is as follows: firstly, adding ethanol into 10mM hydrochloric acid solution to prepare dialysate with ethanol volume fractions of 10%, 20%, 30%, 40% and 50%; then putting the dialysis bag containing the obtained reaction liquid into the dialysate according to the order of decreasing the volume fraction of ethanol in the dialysate, and dialyzing for 3-7 days each time under the condition of stirring at 0 ℃; finally, the dialysis bag containing the obtained reaction solution was dialyzed in a 10mM hydrochloric acid solution for 1 day.
And (3) immersing the dialysis bag containing the reaction solution after methacrylic anhydride removal into a PEG (polyethylene glycol) aqueous solution with the mass concentration of 50% at the temperature of 0 ℃ under normal pressure, and concentrating to obtain a concentrated solution containing the photo-crosslinkable II type collagen.
(3) Preparation of photo-crosslinked type II collagen hydrogel
Under the condition of normal pressure and 0 ℃, the pH value of a concentrated solution containing photo-crosslinkable II collagen is adjusted to 7.5 by using a 5M sodium hydroxide solution, a photoinitiator irgacure2959 is added into the photo-crosslinkable II collagen solution after the pH value is adjusted to prepare a gel pre-polymerizing solution with the photo-crosslinkable II collagen final concentration of 7mg/mL and the photoinitiator final concentration of 1mg/mL, and if the concentration of the gel pre-polymerizing solution after the addition of the photoinitiator is higher and cannot meet the requirements of the photo-crosslinkable II collagen and the photoinitiator final concentration, a corresponding volume of 2M sodium dihydrogen phosphate buffer solution can be supplemented. Then exposing the gel pre-polymerization solution to a strength of 6W/cm2Irradiating for 30 seconds under ultraviolet light to realize the curing of the pre-polymerized liquid and obtain the II type collagen hydrogel.
Example 5
The steps of this example are as follows:
(1) preparing type II collagen solution
Type II collagen was dissolved in a 20mM hydrochloric acid solution at 4 ℃ under normal pressure for 12 hours while stirring the solution to prepare a type II collagen solution having a concentration of 2 mg/mL.
(2) Synthesis of photo-crosslinkable type II collagen
Adjusting the pH value of the II type collagen solution to 7.5 by using 0.2M disodium hydrogen phosphate buffer solution and 5M sodium hydroxide solution under the condition of normal pressure and 4 ℃, then adding methacrylic anhydride into the II type collagen solution after the pH value is adjusted according to the molar ratio of amino groups to methacrylic anhydride in collagen of 1:5, continuously stirring and reacting for 4 hours, and supplementing 5M sodium hydroxide solution in the reaction process to keep the pH value of the reaction system at 7.5.
After the reaction is finished, unreacted methacrylic anhydride in the obtained reaction liquid is removed through dialysis, and the specific mode is as follows: firstly, adding ethanol into a 20mM hydrochloric acid solution to prepare a dialysate with ethanol volume fractions of 10%, 20%, 30%, 40% and 50%; then putting the dialysis bag containing the obtained reaction liquid into the dialysate according to the sequence of the reduction of the volume fraction of the ethanol in the dialysate, and carrying out dialysis for a plurality of times under the condition of stirring at 4 ℃, wherein the dialysis time is 3-7 days each time; finally, the dialysis bag containing the resulting reaction solution was dialyzed in a 20mM hydrochloric acid solution for 2 days.
And (3) removing methacrylic anhydride, and freeze-drying the reaction solution to obtain the photo-crosslinkable II type collagen.
(3) Preparation of photo-crosslinked type II collagen hydrogel
Under the condition of normal pressure and 4 ℃, using 20mM hydrochloric acid solution to dissolve the photo-crosslinkable II collagen and prepare a photo-crosslinkable II collagen solution with the concentration of 10mg/mL, using 5M sodium hydroxide solution to adjust the pH value to 7.0, adding a photoinitiator irgacure2959 into the photo-crosslinkable II collagen solution after the pH value is adjusted to prepare a gel pre-polymerization solution with the final concentration of the photo-crosslinkable II collagen of 4mg/mL and the final concentration of the photoinitiator of 0.5mg/mL, and if the concentration of the gel pre-polymerization solution after the addition of the photoinitiator is higher, the requirements of the final concentrations of the photo-crosslinkable II collagen and the photoinitiator cannot be met, supplementing a corresponding volume of 0.2M disodium hydrogen phosphate buffer solution. The gel prepolymer was then exposed to an intensity of 8W/cm2Irradiating for 30 seconds under ultraviolet light to realize the curing of the pre-polymerized liquid and obtain the II type collagen hydrogel.
In order to detect whether the methacrylic anhydride is successfully grafted to the collagen II, nuclear magnetic resonance analysis is carried out on the type II photocollagen without any treatment and the photocrosslinkable type II collagen obtained in the step (2), and an obtained hydrogen nuclear magnetic resonance image is shown in fig. 1 (b).
Example 6
The steps of this example are as follows:
(1) preparing type II collagen solution
Type II collagen was dissolved in a 1mg/mL 1mM hydrochloric acid solution at 4 ℃ under normal pressure for 12 hours under stirring.
(2) Synthesis of photo-crosslinkable type II collagen
Adjusting the pH value of the II type collagen solution to 8 by using 0.2M sodium dihydrogen phosphate buffer solution and 5M sodium hydroxide solution under the condition of normal pressure and 4 ℃, then adding methacrylic anhydride into the II type collagen solution after the pH value is adjusted according to the molar ratio of amino groups to methacrylic anhydride in collagen of 1:2, continuously stirring and reacting for 6 hours, and supplementing 5M sodium hydroxide solution in the reaction process to keep the pH value of the reaction system at 8.
After the reaction is finished, unreacted methacrylic anhydride in the obtained reaction liquid is removed through dialysis, and the specific mode is as follows: firstly, adding ethanol into 1mM hydrochloric acid solution to prepare dialysate with ethanol volume fractions of 10%, 20%, 30%, 40% and 50%; then putting the dialysis bag containing the obtained reaction liquid into the dialysate according to the sequence of the reduction of the volume fraction of the ethanol in the dialysate, and carrying out dialysis for a plurality of times under the condition of stirring at 4 ℃, wherein the dialysis time is 3-7 days each time; finally, the dialysis bag containing the resulting reaction solution was dialyzed in a 1mM hydrochloric acid solution for 2 days.
And (3) removing methacrylic anhydride, and freeze-drying the reaction solution to obtain the photo-crosslinkable II type collagen.
(3) Preparation of photo-crosslinked type II collagen hydrogel
Under the condition of normal pressure and 4 ℃, 1mM hydrochloric acid is used for dissolving photo-crosslinkable II collagen and preparing a photo-crosslinkable II collagen solution with the concentration of 10mg/mL, the pH value is adjusted to 7.5 by a 5M sodium hydroxide solution, a photoinitiator irgacure2959 is added into the photo-crosslinkable II collagen solution after the pH value is adjusted to prepare a gel pre-polymerization solution with the final concentration of photo-crosslinkable II collagen of 6mg/mL and the final concentration of the photoinitiator of 0.8mg/mL, and if the concentration of the gel pre-polymerization solution after the addition of the photoinitiator is higher, the requirements of the final concentrations of the photo-crosslinkable II collagen and the photoinitiator cannot be met, a corresponding volume of 0.2M sodium dihydrogen phosphate buffer solution can be supplemented. The gel prepolymer was then exposed to an intensity of 8W/cm2Irradiating for 30 seconds under ultraviolet light to realize the curing of the pre-polymerized liquid and obtain the II type collagen hydrogel.
Example 7
The steps of this example are as follows:
(1) preparing type II collagen solution
Type II collagen was dissolved in a 20mM hydrochloric acid solution at 0 ℃ under normal pressure for 12 hours while stirring the solution to prepare a type II collagen solution having a concentration of 2 mg/mL.
(2) Synthesis of photo-crosslinkable type II collagen
Adjusting the pH value of the II type collagen solution to 8 by using 0.2M disodium hydrogen phosphate buffer solution and 5M sodium hydroxide solution under the condition of normal pressure and 0 ℃, then adding methacrylic anhydride into the II type collagen solution after the pH value is adjusted according to the molar ratio of amino groups to methacrylic anhydride in collagen of 1:0.2, continuously stirring and reacting for 4 hours, and supplementing 5M sodium hydroxide solution in the reaction process to keep the pH value of a reaction system at 8.
After the reaction is finished, unreacted methacrylic anhydride in the obtained reaction liquid is removed through dialysis, and the specific mode is as follows: firstly, adding ethanol into a 20mM hydrochloric acid solution to prepare a dialysate with ethanol volume fractions of 10%, 20%, 30%, 40% and 50%; then putting the dialysis bag containing the obtained reaction liquid into the dialysate according to the order of decreasing the volume fraction of ethanol in the dialysate, and dialyzing for 3-7 days each time under the condition of stirring at 0 ℃; finally, the dialysis bag containing the resulting reaction solution was dialyzed in a 20mM hydrochloric acid solution for 2 days.
And (3) removing methacrylic anhydride, and freeze-drying the reaction solution to obtain the photo-crosslinkable II type collagen.
(3) Preparation of photo-crosslinked type II collagen hydrogel
Under the condition of normal pressure and 4 ℃, dissolving photo-crosslinkable II collagen by using 20mM hydrochloric acid, preparing a photo-crosslinkable II collagen solution with the concentration of 10mg/mL, adjusting the pH value to 7.5 by using a 5M sodium hydroxide solution, adding a photoinitiator irgacure2959 into the photo-crosslinkable II collagen solution after the pH value is adjusted to prepare a gel pre-polymerization solution with the final concentration of the photo-crosslinkable II collagen of 4mg/mL and the final concentration of the photoinitiator of 0.5mg/mL, and supplementing a corresponding volume of 0.2M disodium hydrogen phosphate buffer solution if the concentration of the gel pre-polymerization solution after the addition of the photoinitiator is higher and cannot meet the requirements of the final concentrations of the photo-crosslinkable II collagen and the photoinitiator. The gel prepolymer was then exposed to a light intensity of 4W/cm2Irradiating for 60 seconds under ultraviolet light to realize the curing of the pre-polymerized liquid and obtain the II type collagen hydrogel.
Example 8
The steps of this example are as follows:
(1) preparing type II collagen solution
Type II collagen was dissolved in a 20mM hydrochloric acid solution at 4 ℃ under normal pressure for 12 hours while stirring the solution to prepare a type II collagen solution having a concentration of 2 mg/mL.
(2) Synthesis of photo-crosslinkable type II collagen
Adjusting the pH value of the II type collagen solution to 7.5 by using 0.2M disodium hydrogen phosphate buffer solution and 5M sodium hydroxide solution under the condition of normal pressure and 4 ℃, then adding methacrylic anhydride into the II type collagen solution after the pH value is adjusted according to the molar ratio of amino groups to methacrylic anhydride in collagen of 1:5, continuously stirring and reacting for 4 hours, and supplementing 5M sodium hydroxide solution in the reaction process to keep the pH value of the reaction system at 7.5.
After the reaction is finished, unreacted methacrylic anhydride in the obtained reaction liquid is removed through dialysis, and the specific mode is as follows: firstly, adding ethanol into a 20mM hydrochloric acid solution to prepare a dialysate with ethanol volume fractions of 10%, 20%, 30%, 40% and 50%; then putting the dialysis bag containing the obtained reaction liquid into the dialysate according to the sequence of the reduction of the volume fraction of the ethanol in the dialysate, and carrying out dialysis for a plurality of times under the condition of stirring at 4 ℃, wherein the dialysis time is 3-7 days each time; finally, the dialysis bag containing the resulting reaction solution was dialyzed in a 20mM hydrochloric acid solution for 2 days.
And (3) removing methacrylic anhydride, and freeze-drying the reaction solution to obtain the photo-crosslinkable II type collagen.
(3) Preparation of photo-crosslinked type II collagen hydrogel
Under the condition of normal pressure and 4 ℃, using 20mM hydrochloric acid solution to dissolve the photo-crosslinkable II collagen and prepare a photo-crosslinkable II collagen solution with the concentration of 14mg/mL, using 5M sodium hydroxide solution to adjust the pH value to 7.0, adding a photoinitiator irgacure2959 into the photo-crosslinkable II collagen solution after the pH value is adjusted to prepare a gel pre-polymerization solution with the final concentration of the photo-crosslinkable II collagen of 10mg/mL and the final concentration of the photoinitiator of 0.5mg/mL, and if the concentration of the gel pre-polymerization solution after the addition of the photoinitiator is higher, the requirements of the final concentrations of the photo-crosslinkable II collagen and the photoinitiator cannot be met, supplementing a corresponding volume of 0.2M disodium hydrogen phosphate buffer solution. The gel prepolymer was then exposed to an intensity of 8W/cm2Irradiating for 30 seconds under ultraviolet light to realize the curing of the pre-polymerized liquid and obtain the II type collagen hydrogel.
In order to determine whether methacrylic anhydride was successfully grafted onto collagen II, nmr analysis of the photocrosslinkable collagen II without any treatment and the photocrosslinkable collagen II obtained in step (2) of examples 1, 5 and 8 gave a nmr chart as shown in fig. 2, and it can be seen from fig. 2(a) that 5.3 and 5.6ppm (C ═ CH2) and 1.85ppm (CH3) corresponded to the characteristic peaks of the methacrylic anhydride-modified collagen II, indicating that methacrylic anhydride was successfully grafted onto collagen. Similarly, a double bond peak is present between 5ppm and 6ppm in the hydrogen nuclear magnetic resonance image corresponding to the photo-crosslinkable collagen obtained in step (2) of examples 5 and 8 [ as shown in fig. 2(b) and (c) ], which indicates that methacrylic anhydride has been successfully grafted to collagen.
In order to characterize the gel properties of the type II collagen hydrogel provided by the present invention, first, a photograph of the type II collagen hydrogel prepared in example 1 was taken, and as shown in fig. 3(b), it can be seen that the type II collagen hydrogel obtained by the preparation method of the present invention was in a gel state. Meanwhile, DMA (dynamic mechanical analysis) analysis is performed on the collagen II hydrogels prepared in the embodiments 1, 5 and 8, and an obtained storage modulus graph is shown in fig. 3(c), and it can be seen from the graph that the storage modulus of the collagen II hydrogel prepared in the embodiment 1 is about 2 to 3kPa, the storage modulus of the collagen II hydrogel prepared in the embodiment 5 is about 1kPa, and the storage modulus of the collagen II hydrogel prepared in the embodiment 8 is about 6 to 7kPa, which indicates that the collagen II hydrogel obtained by the preparation method provided by the present invention has a certain mechanical strength, and the adjustment of the mechanical strength of the obtained collagen II hydrogel can be realized by adjusting the final concentration of the photocrosslinkable collagen II in the gel pre-polymerization solution.
Application example 1 cell Activity test
The photocrosslinkable type II collagen sample obtained by freeze-drying in the step (2) in the above example 1 is dissolved into a solution of 10mg/mL by using a sterile 20mM hydrochloric acid solution in an ultra-clean bench at normal pressure and at 4 ℃, after the sample is completely dissolved, the pH value is adjusted to 7.0 by using a sterile 5M sodium hydroxide solution, a sterile photoinitiator irgacure2959 is added to prepare a modified type II collagen pre-polymerization solution with the final concentration of type II collagen of 7mg/mL and the final concentration of the photoinitiator of 0.5mg/mL, and if the concentration of the pre-polymerization solution after the photoinitiator is added is higher, a corresponding volume of sterile 0.2 disodium hydrogen phosphate buffer solution can be supplemented.
Digesting the rabbit bone marrow mesenchymal stem cells cultured to the P2 generation by using 1 ml/dish (1 ml trypsin is added to each culture dish) trypsin for 30s, and then uniformly mixing the digested cells with gel prepolymerization liquid, wherein the wrapping cell density is 1 multiplied by 10 for wrapping each milliliter of the prepolymerization liquid7And (4) cells. Then dividing the gel pre-polymerization solution for wrapping the cells into a plurality of groups of samples according to the proportion of each 30 mu L, and exposing each sample to ultraviolet light with the intensity of 8W/cm2Irradiating for 30 seconds to obtain the type II collagen hydrogel wrapping the cells in a three-dimensional way, and after the samples are cultured for 1 day, 4 days, 7 days, 14 days and 21 days respectively, dyeing the samples by FDA/PI solution and imaging by using a laser confocal microscope, wherein the result is shown in figure 4, and the result is shown in figure 4.
Application example 2 cell secretion matrix experiment
The photo-crosslinking collagen sample obtained by freeze-drying in the step (2) in the above example 1 is dissolved into a solution of 10mg/mL by using a sterile 20mM hydrochloric acid solution in an ultra-clean bench under the condition of normal pressure and 4 ℃, after the sample is completely dissolved, the pH value is adjusted to 7.0 by using a sterile 5M sodium hydroxide solution, a sterile photoinitiator irgacure2959 is added to prepare a modified II type collagen pre-polymerization solution with the final concentration of II type collagen of 7mg/mL and the final concentration of the photoinitiator of 0.5mg/mL, and if the concentration of the pre-polymerization solution after the photoinitiator is added is higher, a corresponding volume of sterile 0.2 disodium hydrogen phosphate buffer solution can be supplemented.
Digesting the rabbit bone marrow mesenchymal stem cells cultured to the P2 generation for 30s by using 1 ml/dish trypsin, and then uniformly mixing the digested stem cells with two gel prepolymerization solutions, wherein the wrapping cell density is 1 multiplied by 10 per milliliter of the volume of the prepolymerization solution7And (4) cells. Then dividing the gel pre-polymerization solution for wrapping the cells into a plurality of groups of samples according to the proportion of each 100 mu L, and exposing each sample to ultraviolet light with the intensity of 8W/cm2Irradiating for 30 seconds to obtain II type collagen hydrogel for three-dimensionally wrapping cells, and culturingWas cultured in alpha-MEM medium containing 10% (v/v) serum. After 1 day of in vitro culture, the samples were taken out and implanted subcutaneously into nude mice for in vivo culture. After 14 days, samples were removed and prepared into tissue sections of 10 μm thickness and stained with formulated safranin-O and toluidine blue, as shown in FIG. 5, where a significant deposition of the polysaccharide matrix was observed, indicating that the prepared hydrogel of type II collagen was capable of promoting differentiation of BMSCs towards chondrogenic phase.

Claims (7)

1. A preparation method of II type collagen hydrogel for inducing stem cell chondrogenic differentiation is characterized by comprising the following steps:
(1) preparing type II collagen solution
Dissolving type II collagen in acid solution with pH value of 1-3 at 0-4 deg.C under normal pressure to obtain type II collagen solution with mass concentration of 1-4 mg/mL;
(2) synthesis of photo-crosslinkable type II collagen
Regulating the pH value of the II type collagen solution to be more than 7 and less than or equal to 8 by using a phosphate buffer solution or/and a sodium hydroxide solution at normal pressure and 0-4 ℃, then adding methacrylic anhydride, stirring and reacting for 2-12h at normal pressure and 0-4 ℃, supplementing a sodium hydroxide solution in the reaction process to ensure that the pH value of a reaction system is more than 7 and less than or equal to 8, and metering the adding amount of the methacrylic anhydride according to the molar ratio of amino in the II type collagen solution to the methacrylic anhydride of 1 (0.2-5);
removing unreacted methacrylic anhydride from the obtained reaction liquid after the reaction is finished, then freezing and drying the reaction liquid from which the unreacted methacrylic anhydride is removed to obtain the photocrosslinkable II-type collagen, or filling the reaction liquid from which the unreacted methacrylic anhydride is removed into a dialysis bag, and immersing the dialysis bag containing the reaction liquid into a polyethylene glycol aqueous solution with the mass concentration of 10-50% at the normal pressure and the temperature of 0-4 ℃ for concentrating for 4-24h to obtain a concentrated solution containing the photocrosslinkable II-type collagen;
the unreacted methacrylic anhydride in the obtained reaction solution was removed by dialysis after the reaction was completed in the following manner: firstly, adding ethanol into an acid solution with the pH value of 1-3 to prepare a series of dialyzates, wherein the volume fraction of the ethanol in each dialyzate is sequentially reduced by 10 percent in the series of dialyzates, and the volume fraction of the ethanol in the dialyzate with the largest volume fraction of the ethanol is not more than 50 percent; then putting the dialysis bag containing the reaction solution into the dialysate to perform dialysis for a plurality of times at 0-4 ℃ under stirring, wherein the volume fraction of ethanol in the dialysate replaced by each dialysis is reduced by 10 percent, and the dialysis time is 3-7 days each time; finally dialyzing the dialysis bag containing the reaction solution in an acid solution with the pH value of 2-3 for 1-3 days;
(3) preparation of photo-crosslinked type II collagen hydrogel
Dissolving photo-crosslinkable II type collagen by using an acid solution with the pH value of 1-3 at the normal pressure and the temperature of 0-4 ℃ to obtain a photo-crosslinkable II type collagen solution, adjusting the pH value to 7.0-7.5 by using a sodium hydroxide solution, adding a photoinitiator into the photo-crosslinkable II type collagen solution after the pH value is adjusted to prepare a gel pre-polymerizing solution with the final concentration of the photo-crosslinkable II type collagen of 4-10mg/mL and the final concentration of the photoinitiator of 0.2-1mg/mL, and then placing the gel pre-polymerizing solution under the normal pressure and the room temperature for irradiating for 10-60s under ultraviolet light to obtain the II type collagen hydrogel;
or under the conditions of normal pressure and 0-4 ℃, regulating the pH value of the concentrated solution containing the photocrosslinkable II type collagen to 7.0-7.5 by using a sodium hydroxide solution, adding a photoinitiator into the photocrosslinkable II type collagen solution after the pH value is regulated to prepare a gel pre-polymerization solution with the photocrosslinkable II type collagen final concentration of 4-10mg/mL and the photoinitiator final concentration of 0.2-1mg/mL, and then placing the gel pre-polymerization solution under ultraviolet light at normal pressure and room temperature for irradiating for 10-60s to obtain the II type collagen hydrogel.
2. The method for preparing type II collagen hydrogel for inducing chondrogenic differentiation of stem cells according to claim 1, wherein in the step (3), the photoinitiator is irgacure2959, irgacure184, irgacure127, irgacure500, irgacure651 or rose Bengal.
3. The method for preparing type II collagen hydrogel for inducing stem cell chondrogenic differentiation according to claim 1, wherein a gel pre-polymerization solution satisfying the requirements of the final concentration of photocrosslinkable type II collagen and the final concentration of the photoinitiator is obtained by supplementing a phosphate buffer solution after the photoinitiator is added in the step (3).
4. The method for preparing type II collagen hydrogel for inducing chondrogenic differentiation of stem cells according to claim 3, wherein the ultraviolet light intensity in the step (3) is 4 to 8W/cm2。
5. The method for preparing type II collagen hydrogel for inducing chondrogenic differentiation of stem cells according to claim 1, wherein the molar concentration of the sodium hydroxide solution in the steps (2) and (3) is 0.5 to 5M.
6. The method for preparing type II collagen hydrogel for inducing chondrogenic differentiation of stem cells according to claim 3, wherein the phosphate buffer solution is used in the step (2) and the step (3) at a molar concentration of 0.2 to 2M; the phosphate buffer solution is disodium hydrogen phosphate solution or sodium dihydrogen phosphate solution.
7. The method for preparing type II collagen hydrogel for inducing chondrogenic differentiation of stem cells according to claim 1, wherein the acid solution in the steps (1), (2) and (3) is hydrochloric acid solution or acetic acid solution.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120171258A1 (en) * 2009-06-12 2012-07-05 Sefton Michael V Modular Assembly of Tissue Engineered Constructs
CN104788583A (en) * 2014-01-17 2015-07-22 北京朗嘉仪生物技术有限公司 Photo-crosslinking material, hydrogel formed by photo-crosslinking material and preparation method and application thereof
CN105936671A (en) * 2016-06-06 2016-09-14 陕西科技大学 Preparation method of vinyl collagen

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