Disclosure of Invention
The invention aims to provide a preparation method of printable composite hydrogel with high toughness, aiming at the defects that the traditional hydrogel bracket has insufficient toughness, cannot induce and regulate the cell specific differentiation phenotype and the manufacturing technology cannot realize individuation and complex geometric shapes. The repair scaffold prepared from the hydrogel obtained by the preparation method has good biocompatibility and cartilage differentiation induction capability, and compared with gelatin-based hydrogel, the mechanical strength of the repair scaffold is obviously improved. The invention improves the mechanical property and mechanical anisotropy of the hydrogel through nano covalent compounding/hydrogen bonds and regulates the cell proliferation and differentiation.
Another object of the present invention is to provide printable composite hydrogels with high toughness obtained by the above preparation method.
It is a further object of the present invention to provide the use of the printable composite hydrogel with high toughness described above.
The purpose of the invention is realized by the following technical scheme: a preparation method of printable composite hydrogel with high toughness comprises the following steps of modifying gelatin and cellulose nanocrystals respectively, wherein the modified gelatin and the modified cellulose nanocrystals are formed by nano covalent compounding/hydrogen bond double crosslinking, so that the printable composite hydrogel with high toughness is obtained:
(1) preparation of modified gelatin: dissolving gelatin in water to obtain gelatin solution; adjusting the pH value of a gelatin solution to 8-9, then adding a monomer containing a methacryloyl group to obtain a reaction solution A, reacting, and controlling the pH value of the reaction solution A to be 8-9 in the reaction process; putting the obtained reaction in a dialysis bag for dialysis and drying to obtain modified gelatin;
(2) preparing modified cellulose nanocrystals: treating the cellulose nanocrystal by adopting an N-methylmorpholine oxide (NMMO) ionic liquid system or a tetramethylpiperidine oxynitride (TEMPO) catalytic oxidation system; putting the obtained reaction product into a dialysis bag for dialysis and drying to obtain modified cellulose nanocrystalline;
(3) preparation of composite hydrogel: dissolving the modified gelatin prepared in the step (1) with water to obtain a modified gelatin solution; adding the modified cellulose nanocrystal prepared in the step (2), N-hydroxysuccinimide (NHS) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) to obtain a reaction liquid C, reacting, dialyzing and drying to obtain printable composite hydrogel with high toughness; wherein the dosage of each substance in the reaction liquid C is as follows by mass percent: 5-20% of modified gelatin, 0.2-2% of modified cellulose nanocrystal, N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride as catalytic agents, and the balance of water.
The concentration of the gelatin solution in the step (1) is preferably 5-20% by mass; more preferably 10% by mass.
The gelatin described in step (1) is preferably a gelatin derived from bovine skin.
The gelatin in the step (1) is preferably gelatin with the molecular weight of 3-10 ten thousand.
The monomer containing a methacryl group in the step (1) is preferably one or both of methacrylic anhydride and glycidyl methacrylate.
The amount of the methacryl group-containing monomer described in step (1) is preferably such that the ratio of the methacryl group-containing monomer: gelatin (1-2) mL: 2g, calculating; more preferably, the ratio of the monomer containing a methacryl group: gelatin ═ 1.5 mL: and 2 g.
The pH value in the step (1) is preferably adjusted by sodium hydroxide; more preferably by means of a NaOH solution having a concentration of 5 mol/L.
The reaction temperature in the step (1) is preferably 20-50 ℃; more preferably 40 deg.c.
The reaction time in the step (1) is preferably 3-8 h; more preferably 6 h.
The dialysis bag in the step (1) is preferably a dialysis bag with molecular weight cutoff of 3500.
The dialysis time in the step (1) is preferably 48-96 h; more preferably 72 h.
The cellulose nanocrystal in the step (2) is preferably a cellulose nanocrystal with the length-diameter ratio of more than 150.
Step (2) is preferably: uniformly mixing cellulose nanocrystals, NaBr, tetramethylpiperidine oxynitride (TEMPO), NaClO and water to obtain a reaction liquid B, and adjusting the pH value of the reaction liquid B to 10-10.5; uniformly dispersing and then reacting; after the reaction is finished, adjusting the pH value to 6.8-7.2, and dialyzing to obtain modified cellulose nanocrystals; wherein the dosage of each substance in the reaction liquid B is as follows: NaBr: TEMPO: NaClO is 1: (0.2-0.3): (0.02-0.03): (0.2-0.4).
The dosage of each substance in the reaction liquid B is more preferably as follows: NaBr: TEMPO: NaClO is 1: 0.25: 0.025: 0.3 proportion.
The dispersion is preferably by ultrasonic dispersion.
The ultrasonic wave is preferably treated at 25kHz and 1000W for 5 min.
The reaction time is preferably 30-80 min; more preferably 60 min.
The reaction temperature is preferably 20-30 ℃.
The pH value of the reaction liquid B is preferably adjusted to 10-10.5 by using alkali, and then buffer solution is added to fix the volume to the reaction volume, so that the pH value of the reaction liquid B is stable in the reaction process.
The buffer solution is preferably Na with the pH value of 10-10.52CO3-NaHCO3And (4) buffer dissolving.
The pH value after the reaction is preferably adjusted by HCl solution.
The dialysis is preferably performed by using a dialysis bag with a cut-off of 12000.
The dialysis time is preferably 5 days.
The concentration of the modified gelatin solution in the step (3) is preferably 5-20% by mass/volume ratio.
The N-hydroxysuccinimide and the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in the step (3) are preferably mixed in a mass ratio of 1: 2, proportioning.
The dosage of the catalyst in the step (3) is preferably as follows: catalyst 0.2%: and 45 mg. Namely, the final concentration of the modified cellulose nanocrystal is 0.2%, and the catalyst is prepared in an amount of 45 mg.
The dialysis described in the step (3) is preferably performed by using a dialysis bag having a molecular weight cut-off of 3500.
Printable composite hydrogel with high toughness is obtained by the preparation method. The hydrogel has good toughness and strength and good biocompatibility, and can be used for 3D printing.
The printable composite hydrogel with high toughness is applied to preparation of biomedical engineering materials.
The biomedical engineering material is preferably a biomedical engineering material for soft tissue repair.
A biomedical engineering material is prepared by the following steps: preparing the printable composite hydrogel with high toughness into a hydrogel solution, and adding a photoinitiator to obtain a reaction solution D; adding or not adding functional substances according to the use purpose, and carrying out photocuring molding to obtain the biomedical engineering material.
The solvent in the hydrogel solution comprises water, buffer solution and cell culture medium; preferably cell culture medium, more preferably DMEM-F12 medium.
The concentration of the printable composite hydrogel with high toughness in the hydrogel solution is 8-15% by mass volume ratio.
The photoinitiator is preferably Irgacure series photoinitiator; more preferably the photoinitiator Irgacure 2959.
The dosage of the photoinitiator is preferably calculated according to the mass volume ratio of the concentration of the photoinitiator in the reaction liquid D of 0.03-0.06%; more preferably 0.05% by mass/volume.
The functional substance is preferably a cell growth factor and/or a stem cell.
The stem cell is preferably a human adipose-derived mesenchymal stem cell.
The addition amount of the stem cells is preferably 106~108The amount of individual stem cells per mL of reaction solution D; more preferably according to 107The amount of individual stem cells per mL of reaction solution D was added.
The light curing molding condition is light curing under ultraviolet light; preferably, the light curing is carried out under the ultraviolet light of 360-370 nm; more preferably, it is photocured under 365nm ultraviolet light.
The photocuring forming time is 1-10 min; preferably for 5 min.
The photocuring forming is preferably realized by a 3D printer.
The 3D printer is a 3D printer adopting the working principle of a stereo Stereolithography (SLA), and is preferably a 3D printer of a 3D systems ProX 800 model.
The biomedical engineering material is applied to tissue repair; is particularly suitable for being applied to soft tissue repair; is more suitable for application in cartilage repair.
Compared with the prior art, the invention has the following advantages and effects:
(1) in earlier researches, the inventor finds that the mechanical properties of the scaffold material can influence the migration, proliferation and differentiation expression of stem cells, and the nano-sized fibers are helpful for the differentiation of the cells to cartilage. The inventor modifies gelatin to enable the hydrogel material to have photocuring capacity and printability, and modifies nanocrystalline cellulose to obtain a stronger covalent bond and hydrogen bond double-crosslinking network between the modified cellulose nanocrystal and the modified gelatin, so that the mechanical strength of the hydrogel is greatly improved, and the dispersibility of the modified nanocrystal is better. The combination of the above advantages helps the differentiation and expression of stem cells to cartilage.
(2) The hydrogel support provided by the invention is prepared by a photocuring 3D printing technology, the high-toughness hydrogel three-dimensional space and nontoxicity obviously improve the cell survival rate, and the cell survival rate is far higher than that of hydrogel prepared by unmodified gelatin and modified cellulose nanocrystals in other modes.
(3) The invention can print out complicated structures such as vascular forks, heart valves and the like and more complicated tissues and organs which meet the clinical requirements, and has wider application.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Example 1
(1) Preparing modified gelatin and modified cellulose nanocrystals:
preparation of modified gelatin: weighing 10G of gelatin (Sigma, G9382 from cow leather) and dissolving in 100mL of ultrapure water to prepare a gelatin solution with the mass concentration of 10%, and then adjusting the pH to 8.5 by using a 5mol/L NaOH solution; dropwise adding 7.5mL of methacrylic anhydride, reacting for 6h at 40 ℃, and controlling the pH of the reaction solution to be 8-9 through NaOH in the whole reaction process; and (3) putting the product obtained by the reaction into a dialysis bag with the molecular weight cutoff of 3500 for dialysis for 3 days, and freeze-drying to obtain the modified gelatin.
Preparing modified cellulose nanocrystals: 1g of Cellulose nanocrystals (12% Cellulose nanocrystal suspension, pore size distribution, 90-300nm, crystallization index 87%, available from Cellulose lab, Canada) were weighed out, 0.25g of NaBr, 0.025g of TEMPO and 2.5mL of a 12% strength by weight NaClO solution were addedNaOH is used for adjusting the pH value to 10-10.5. Using Na with pH value of 10.22CO3-NaHCO3Diluting the buffer solution to 200mL, treating for 5min by ultrasonic waves (25kHz, ultrasonic wave power 1000W), reacting for 60min at normal temperature, and then adding 10mL of absolute ethyl alcohol to terminate the reaction. Adjusting pH to 7 with 0.1mol/L HCl solution, dialyzing with dialysis bag with cut-off molecular weight of 12000 for 5 days to obtain modified cellulose nanocrystal, and storing in refrigerator at 4 deg.C.
(2) Preparation of composite hydrogel: preparing 50mL of modified gelatin with the concentration of 8% by mass by using ultrapure water, adding the modified cellulose nanocrystal prepared in the step (1) to enable the final mass ratio to be 0.2%, adding 15mg of N-hydroxysuccinimide (NHS), shaking uniformly, adding 30mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), reacting at normal temperature for 15min, transferring into a dialysis bag with the molecular weight cutoff of 3500 for dialysis for 5 days, and freeze-drying in a refrigerator at-20 ℃ for later use.
(3) Preparation of cell-loaded hydrogel: sterilizing all materials for more than 1h under ultraviolet, and dissolving the composite hydrogel prepared in the step (2) in a DMEM-F12 culture medium to obtain a solution A, wherein the concentration of the composite hydrogel in the solution A is 8% by mass-volume ratio; to the solution A was added a solution of Irgacure2959 (CIBA Chemicals) as a photoinitiator to obtain a solution B in which the final concentration of Irgacure2959 was 0.05% by mass/volume. Press 107Amount of cells/mL solution B, human adipose-derived mesenchymal stem cells (HADMSC cells, Sigma) were seeded into solution B to obtain solution C. And injecting the solution C into a disc mold with the diameter of 8mm and the thickness of 3mm, and then placing the disc mold under 365nm ultraviolet light for photocuring for 5min to obtain the high-toughness composite hydrogel with cartilage differentiation capability. When a specific shape is required, the solution C can be printed and formed by a 3D systems ProX 800 and photocured in the printing process to obtain the composite hydrogel scaffold with high toughness and cartilage differentiation capacity.
(4) Photocuring of cell-free hydrogels: dissolving the composite hydrogel prepared in the step (2) in deionized water to prepare 8% hydrogel solution, adding a photoinitiator Irgacure2959 (CIBA Chemicals) solution, and enabling the final concentration of the Irgacure2959 to be 0.05% by mass and volume. And (3) injecting the composite hydrogel solution into a disc die with the diameter of 8mm and the thickness of 3mm, and then placing the disc die under 365nm ultraviolet light for photocuring for 5min to obtain the high-toughness composite hydrogel for testing mechanical properties and calculating strain energy.
Example 2
(1) Modified gelatin and modified cellulose crystals were prepared as in example 1.
(2) Preparation of composite hydrogel: preparing 50mL of modified gelatin with the concentration of 8% by mass by using ultrapure water, adding the modified cellulose nanocrystal prepared in the step (1) to enable the final mass ratio to be 0.8%, adding 60mg of NHS, shaking up, adding 120mg of EDC, reacting for 15min at normal temperature, transferring into a dialysis bag (molecular weight of 3500), dialyzing for 5 days, and freeze-drying in a refrigerator at-20 ℃ for later use.
(3) Preparation of cell-loaded hydrogel: sterilizing all materials for more than 1h under ultraviolet, and dissolving the composite hydrogel in a DMEM-F12 culture medium to obtain a solution A, wherein the concentration of the composite hydrogel is 8% by mass and volume; to the solution A was added a solution of Irgacure2959 (CIBA Chemicals) as a photoinitiator to obtain a solution B in which the final concentration of Irgacure2959 was 0.05% by mass/volume. Press 107Amount of cells/mL solution B, human adipose-derived mesenchymal stem cells (HADMSC cells, Sigma) were seeded into solution B to obtain solution C. And injecting the solution C into a disc mold with the diameter of 8mm and the thickness of 3mm, and then placing the disc mold under 365nm ultraviolet light for photocuring for 5min to obtain the high-toughness composite hydrogel with cartilage differentiation capability. When a specific shape is required, the solution C can be printed and formed by a 3D systems ProX 800 and photocured in the printing process to obtain the composite hydrogel scaffold with high toughness and cartilage differentiation capacity.
(4) Photocuring of cell-free hydrogels: dissolving the composite hydrogel prepared in the step (2) in deionized water to prepare 8% hydrogel solution, adding a photoinitiator Irgacure2959 (CIBA Chemicals) solution, and enabling the final concentration of the Irgacure2959 to be 0.05% by mass and volume. And (3) injecting the composite hydrogel solution into a disc die with the diameter of 8mm and the thickness of 3mm, and then placing the disc die under 365nm ultraviolet light for photocuring for 5min to obtain the high-toughness composite hydrogel for testing mechanical properties and calculating strain energy.
Example 3
(1) Modified gelatin and modified cellulose crystals were prepared as in example 1.
(2) Preparation of composite hydrogel: preparing 50mL of modified gelatin with the concentration of 8% by mass by using ultrapure water, adding the modified cellulose nanocrystal prepared in the step (1) to enable the final mass ratio to be 2.0%, adding 150mg of NHS, shaking up, adding 300mg of EDC, reacting for 15min at normal temperature, transferring into a dialysis bag (molecular weight of 3500), dialyzing for 5 days, and freeze-drying in a refrigerator at-20 ℃ for later use.
(3) Preparation of cell-loaded hydrogel: sterilizing all materials for more than 1h under ultraviolet, and dissolving the composite hydrogel in a DMEM-F12 culture medium to obtain a solution A, wherein the concentration of the composite hydrogel is 8% by mass and volume; to the solution A was added a solution of Irgacure2959 (CIBA Chemicals) as a photoinitiator to obtain a solution B in which the final concentration of Irgacure2959 was 0.05% by mass/volume. Press 107Amount of cells/mL solution B, human adipose-derived mesenchymal stem cells (HADMSC cells, Sigma) were seeded into solution B to obtain solution C. And injecting the solution C into a disc mold with the diameter of 8mm and the thickness of 3mm, and then placing the disc mold under 365nm ultraviolet light for photocuring for 5min to obtain the high-toughness composite hydrogel with cartilage differentiation capability. When a specific shape is required, the solution C can be printed and formed by a 3D systems ProX 800 and photocured in the printing process to obtain the composite hydrogel scaffold with high toughness and cartilage differentiation capacity.
(4) Photocuring of cell-free hydrogels: dissolving the composite hydrogel prepared in the step (2) in deionized water to prepare 8% hydrogel solution, adding a photoinitiator Irgacure2959 (CIBA Chemicals) solution, and enabling the final concentration of the Irgacure2959 to be 0.05% by mass and volume. And (3) injecting the composite hydrogel solution into a disc die with the diameter of 8mm and the thickness of 3mm, and then placing the disc die under 365nm ultraviolet light for photocuring for 5min to obtain the high-toughness composite hydrogel for testing mechanical properties and calculating strain energy.
Example 4
(1) The modified gelatin and modified cellulose nanocrystals were prepared as in example 1.
(2) Preparation of composite hydrogel: preparing 50mL of modified gelatin with the concentration of 20% by mass by using ultrapure water, adding the modified cellulose nanocrystal prepared in the step (1) to enable the final mass ratio to be 1.5%, adding 100mg of NHS, shaking up, adding 200mg of EDC, reacting at normal temperature for 15min, transferring into a dialysis bag (molecular weight of 3500) for dialysis for 5 days, changing water every day, and freeze-drying in a refrigerator at-20 ℃ for later use.
(3) Preparation of cell-loaded hydrogel: sterilizing all materials for more than 1h under ultraviolet, and dissolving the composite hydrogel in a DMEM-F12 culture medium to obtain a solution A, wherein the concentration of the composite hydrogel is 8% by mass and volume; to the solution A was added a solution of Irgacure2959 (CIBA Chemicals) as a photoinitiator to obtain a solution B in which the final concentration of Irgacure2959 was 0.05% by mass/volume. Press 107Amount of cells/mL solution B, human adipose-derived mesenchymal stem cells (HADMSC cells, Sigma) were seeded into solution B to obtain solution C. And injecting the solution C into a disc mold with the diameter of 8mm and the thickness of 3mm, and then placing the disc mold under 365nm ultraviolet light for photocuring for 5min to obtain the high-toughness composite hydrogel with cartilage differentiation capability. When a specific shape is required, the solution C can be printed and formed by a 3D systems ProX 800 and photocured in the printing process to obtain the composite hydrogel scaffold with high toughness and cartilage differentiation capacity.
Example 5
(1) The modified gelatin and modified cellulose nanocrystals were prepared as in example 1.
(2) Preparation of composite hydrogel: preparing 50mL of modified gelatin with the concentration of 5% by mass by using ultrapure water, adding the modified cellulose nanocrystal prepared in the step (1) to enable the final mass ratio to be 0.5%, adding 30mg of NHS, shaking up, adding 60mg of EDC, reacting at normal temperature for 15min, transferring into a dialysis bag (molecular weight of 3500) for dialysis for 5 days, changing water every day, and freeze-drying in a refrigerator at-20 ℃ for later use.
(3) Preparation of cell-loaded hydrogel: sterilizing all materials for more than 1h under ultraviolet, and dissolving the composite hydrogel in a DMEM-F12 culture medium to obtain a solution A, wherein the concentration of the composite hydrogel is 15% by mass and volume; adding light to solution ASolution of Irgacure2959 (CIBA Chemicals) as an initiator gave solution B, in which the final concentration of Irgacure2959 was 0.05% by mass/volume. Press 107Amount of cells/mL solution B, human adipose-derived mesenchymal stem cells (HADMSC cells, Sigma) were seeded into solution B to obtain solution C. And injecting the solution C into a disc mold with the diameter of 8mm and the thickness of 3mm, and then placing the disc mold under 365nm ultraviolet light for photocuring for 5min to obtain the high-toughness composite hydrogel with cartilage differentiation capability. When a specific shape is required, the solution C can be printed and formed by a 3D systems ProX 800 and photocured in the printing process to obtain the composite hydrogel scaffold with high toughness and cartilage differentiation capacity.
Comparative example 1
The modified gelatin was prepared as in example 1.
Preparation of cell-loaded hydrogel: all materials are sterilized for more than 1h under ultraviolet, the modified gelatin hydrogel is dissolved in DMEM-F12 culture medium according to the mass volume ratio of 8%, and a photoinitiator Irgacure2959 (CIBA Chemicals company) solution is added into the solution A to obtain a solution B, wherein the final concentration of the Irgacure2959 is 0.05% by mass volume ratio. Press 107Amount of cells/mL solution B, human adipose-derived mesenchymal stem cells (HADMSC cells, Sigma) were seeded into solution B to obtain solution C. And injecting the solution C into a disc mold with the diameter of 8mm and the thickness of 3mm, and then placing under 365nm ultraviolet light for photocuring for 5min to obtain the cell-loaded hydrogel.
Photocuring of cell-free hydrogels: the modified gelatin is dissolved in deionized water to prepare 8% hydrogel solution, and a photoinitiator Irgacure2959 (CIBA Chemicals) solution is added to make the final concentration of the Irgacure2959 be 0.05% by mass and volume. And (3) injecting the composite hydrogel solution into a disc die with the diameter of 8mm and the thickness of 3mm, and then placing the disc die under 365nm ultraviolet light for photocuring for 5min to obtain the high-toughness composite hydrogel for testing mechanical properties and calculating strain energy.
Comparative example 2
(1) The modified gelatin was prepared as in example 1.
(2) Preparation of composite hydrogel: preparing 50mL of modified gelatin with the concentration of 8% by mass by using ultrapure water, adding unmodified cellulose nanocrystal to enable the final mass ratio to be 0.8%, adding 60mg of NHS, shaking uniformly, adding 120mg of EDC, reacting for 15min at normal temperature, transferring into a dialysis bag (molecular weight of 3500), dialyzing for 5 days, and freeze-drying in a refrigerator at-20 ℃ for later use.
(3) Preparation of cell-loaded hydrogel: all materials are sterilized under ultraviolet for more than 1h, the composite hydrogel prepared in the step (2) is dissolved in DMEM-F12 culture medium according to the proportion of 8%, and a photoinitiator Irgacure2959 (CIBA Chemicals) solution is added into the solution A to obtain a solution B, wherein the final concentration of the Irgacure2959 is 0.05 percent of the mass-to-volume ratio. Press 107Amount of cells/mL solution B, human adipose-derived mesenchymal stem cells (HADMSC cells, Sigma) were seeded into solution B to obtain solution C. And injecting the solution C into a disc mold with the diameter of 8mm and the thickness of 3mm, and then placing the disc mold under 365nm ultraviolet light for photocuring for 5min to obtain the high-toughness composite hydrogel with cartilage differentiation capability.
(4) Photocuring of cell-free hydrogels: dissolving the composite hydrogel prepared in the step (2) in deionized water to prepare 8% hydrogel solution, adding a photoinitiator Irgacure2959 (CIBA Chemicals) solution, and enabling the final concentration of the Irgacure2959 to be 0.05% by mass and volume. And (3) injecting the composite hydrogel solution into a disc die with the diameter of 8mm and the thickness of 3mm, and then placing the disc die under 365nm ultraviolet light for photocuring for 5min to obtain the high-toughness composite hydrogel for testing mechanical properties and calculating strain energy.
Comparative example 3
(1) Preparing dialdehyde nano cellulose (DAC): the mass ratio of the nano-cellulose to the sodium periodate is 1:1, the reaction is carried out for 6 hours at 40 ℃ in a dark place, and then the solution is centrifuged and dialyzed until free ions in the solution are completely removed;
(2) preparation of gelatin solution: adding gelatin into Phosphate Buffer Solution (PBS), and continuously stirring until the gelatin is completely dissolved;
(3) preparation of cell-free dialdehyde nano cellulose (DAC)/Gelatin (GEL) composite hydrogel: mixing a 4% DAC solution and a 4% GEL solution in a volume ratio of 1:1, quickly stirring uniformly, injecting into a disc mold with the diameter of 8mm and the thickness of 3mm, and incubating at 37 ℃ for 2 hours to obtain the composite hydrogel.
(4) Preparation of cell-loaded hydrogel: sterilizing all materials for more than 1h under ultraviolet, uniformly mixing human adipose-derived mesenchymal stem cells (HADMSC cells, Sigma) and a GEL solution with the concentration of 4%, then adding a DAC solution with the concentration of 4%, rapidly and uniformly stirring, injecting into a disc mold with the diameter of 8mm and the thickness of 3mm, and incubating for 2 hours at 37 ℃ to obtain a composite hydrogel; wherein, the DAC solution with the concentration of 4 percent and the GEL solution with the concentration of 4 percent are mixed according to the volume ratio of 1:1 to form a solution B, and the addition amount of the human adipose-derived mesenchymal stem cells is 107The amount of cells per mL of solution B was calculated.
Effects of the embodiment
(1) The cell-free hydrogels prepared in the above comparative examples 1 to 3 and examples 1 to 3 were subjected to a stress strain test by a compression model using a dynamic thermomechanical analyzer, and then to a strain energy calculation formula according to the formula
And calculating to obtain the strain energy. The results of the test experiments are shown in fig. 1, and it is apparent that the strain energy of examples 1 to 3 is much higher than that of comparative examples 1 to 3.
(2) The experimental procedure of laser confocal scanning microscope observation after culturing the hydrogels loaded with HADMSC of examples 1 to 3 and comparative examples 1 to 3 in DMEM F12 culture medium for 14 days: each sample was prepared by placing a disc of hydrogel in one well of a 24-well plate, adding 0.5mL of prepared staining solution (the staining solution ratio was 1. mu.L of Calcein, 1. mu.L of ethidium bromide dimer (ethidium homomodimer-1, EthD-1) to each well, and placing in a chamber at 37 ℃ in CO2And (5) culturing in an incubator for 30min, and observing the growth condition of the cells by using a laser confocal scanning microscope. The results are shown in fig. 2, and it can be seen that all groups of cells grew well on the hydrogel scaffold, and examples 1-3 showed better cell migration with increasing content of modified cellulose nanocrystals relative to the control group.
(3) After the hydrogels of examples 1-3 and comparative examples 1-3 loaded with HADMSC were cultured in DMEM F12 medium for 14 days, Aggrecan (Aggrecan) and transcription factor sox9 were tested for cartilage related characteristic genes, and the results are shown in FIG. 3. The detection steps are as follows:
separating and purifying RNA: taking one hydrogel disk sheet (with the diameter of 8mm and the thickness of 3mm) after being cultured for 14 days, adding 0.5mL of TRIzol, mashing, standing for 5min, adding 0.1mL of chloroform, uniformly mixing, standing for 2min, centrifuging at 12000rcf at 4 ℃ for 15min, transferring upper-layer aqueous phase RNA to a new centrifuge tube, adding 0.5mL of isopropanol containing glycogen blue, mixing, standing for 10min, centrifuging at 10000rcf at 4 ℃ for 10min, removing supernatant, adding 0.7mL of 75% ethanol, uniformly mixing, centrifuging at 7500rcf at 4 ℃ for 5min, removing supernatant, naturally airing for 20min, adding 20 mu L of nuclease-free water, beating, and detecting the purity by using Nanodrop 2000;
② synthesizing cDNA: preparing a reaction solution containing qScript, adding the extracted RNA, and then completing the synthesis and cloning of cDNA on an Eppendorf Mastercycler device;
③ qPCR: preparing an upstream primer and a downstream primer containing 18s, Aggrecan and sox9 genes and SYBR reaction solution, adding the cDNA obtained in the step II into the reaction solution, sealing a membrane, centrifuging for 10s, and finishing qPCR in a Bio-rad CFX connect Real time system, wherein the adopted PCR reaction system is as follows:
mu.L of nuclease-free water, 1. mu. L, SYBR 10 each of the upstream primer at a concentration of 10. mu.M and the downstream primer at a concentration of 10. mu.M, and 2. mu.L of the template cDNA (synthesized in the second step).
The PCR reaction conditions are as follows: denaturation at 95 deg.C for 5 min; 30 cycles of 94 ℃ for 45s, 55 ℃ for 45s and 72 ℃ for 60 s; extension at 72 ℃ for 10 min.
18s upstream primer: 5'-CCAACCTGGTTGATCCTGCCAGTA-3', respectively;
18s downstream primer: 5'-CCTTGTTAACGACTTCACCTTCCTCT-3', respectively;
aggrecan upstream primer: 5'-ATGCCCAAGACTACCAGTGG-3', respectively;
aggrecan downstream primer: 5'-TCCTGGAAGCTCTTCTCAGT-3', respectively;
sox9 upstream primer: 5'-TCCTCAGGCTTTGCGATTT-3', respectively;
sox9 downstream primer: 5'-TGCTCGGGCACTTATTGG-3' are provided.
From the expression results of these two genes, both were significantly expressed in the composite hydrogel group (examples 1 to 3) after 14 days of culture, compared to the control group (comparative examples 1 to 3), which means that the composite hydrogel contributes to the differentiation expression of stem cells into cartilage.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
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