CN110256856B - Injectable gelatin-sodium alginate porous cryogel with shape memory and preparation method thereof - Google Patents
Injectable gelatin-sodium alginate porous cryogel with shape memory and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an injectable gelatin-sodium alginate porous cryogel with shape memory and a preparation method thereof. The invention improves the defects of poor biocompatibility, poor mechanical property and difficult realization of injectability of the existing cryogel by simulating the structure of the natural extracellular matrix. The penetration rate of the frozen gel material prepared by the optimization method can reach 17-43 percent, and the swelling rate in 2 hours can reach 7.4-10.6.
Description
Technical Field
The invention belongs to the field of biomedical tissue engineering materials, and particularly relates to an injectable gelatin-sodium alginate porous cryogel with shape memory and a preparation method thereof.
Background
The tissue engineering is that seed cells (cultured cells or stem cells) are planted in an artificial extracellular matrix composition structure, are cultured and expanded in vitro to form new tissues, and then are implanted into a patient body to be integrated with the tissues so as to regenerate and rebuild the tissues. The artificial three-dimensional scaffold used in tissue engineering should simulate the composition and function of the extracellular matrix of natural tissue as much as possible. In tissue engineering strategies, injectable in situ-forming hydrogels have the following advantages over preformed scaffolds: the initial sol/cell suspension can fill any shape of defect and can be mixed with various therapeutic drugs, implanted without surgery, etc. Therefore, the in-situ formed gel has wide application prospect in tissue engineering.
Currently, injectable tissue engineering is mostly delivered in liquid form by prepolymers of injectable hydrogels, requiring that the prepolymers must be able to flow through small bore needles or medical catheters and then gel in the diseased area. However, this method has many disadvantages, firstly, it is difficult to determine the gel time according to different diseased regions, and secondly, this method cannot ensure the formation of a gel system with proper mechanical strength maintenance and in vivo stability; furthermore, this approach does not guarantee the maintenance of protein, drug or cellular activity in the harsh environment in vivo; finally, the gel liquid prepolymer is likely to leak into adjacent tissues or be diluted by body fluids during re-injection, which may affect not only the gelling process of the hydrogel but also the physicochemical structure of the final gel.
Cryogels are unique scaffold materials created by the controlled freezing/thawing of polymer solutions. The frozen state is the main stage of the synthesis of the cryogel, the chemical reaction is carried out between minus 5 ℃ and minus 20 ℃, the prepolymer is physically crosslinked or chemically crosslinked to gel, and most of the solvent is crystallized to induce the gelation of the hydrogel. These solvent crystals act as porogens, while the hydrogel component keeps the liquid phase wrapped around the solvent crystals. The freezing rate has a great effect on freezing the gel, where a slower rate may result in larger pores and increased interconnectivity of the gel. And thawing at room temperature after freezing for a proper time, melting crystals, and forming an interconnected macroporous structure in the gel. Compared with the traditional hydrogel, the cold gel usually shows enhanced mechanical stability and an ideal porous structure, and is a very potential tissue engineering scaffold material. Currently, cryogels can be used for the expansion and loading of various cells and the construction of delivery systems for related signaling molecules or drugs in the field of tissue engineering. Unlike most in situ gel systems, the solid injectable jelly has excellent in vivo stability and interconnected macroporous structure, and has more advantages in promoting cell infiltration and transportation capacity compared with the hydrogel scaffold with a nano-porous structure, so that the solid injectable jelly is a very potential injectable tissue engineering scaffold material.
Therefore, research and exploration of cryogels as biomedical tissue engineering scaffold materials are important research subjects for those skilled in the art.
Disclosure of Invention
In view of the above, the present invention aims to prepare a porous injectable cryogel with natural extracellular matrix collagen and proteoglycan components and a loose porous structure and a shape memory function, so as to deliver a completely preformed cryogel to a lesion region through a conventional needle injector, complete tissue defect repair, and improve the problems of the existing injectable tissue engineering scaffold material.
In order to realize the aim, the invention discloses an injectable gelatin-sodium alginate porous cryogel with shape memory and a preparation method thereof.
The specific technical scheme of the invention is as follows:
1. a preparation method of injectable gelatin-sodium alginate porous cryogel with shape memory comprises the following steps:
1) mixing and dissolving methacrylic anhydride modified gelatin and sodium alginate in a PBS solution at the temperature of 25-60 ℃, and recording the mixed solution A;
2) further sequentially adding tetramethylethylenediamine and an ammonium persulfate solution into the mixed solution A obtained in the step 1), uniformly mixing, and recording a mixed solution B;
3) sucking the mixture B obtained in the step 2) into a mould in time, and freezing for 18-24 hours at-20 to-80 ℃ to prepare the gelatin-sodium alginate porous cryogel.
Preferably, the preparation method of the methacrylic anhydride modified gelatin in the step 1) comprises the following steps: weighing 5g of A type gelatin, dissolving the gelatin in a 50mL PBS solution, heating the gelatin to 50 ℃ by using a magnetic stirrer, stirring the gelatin at a constant speed, slowly dripping 5mL of methacrylic anhydride solution after complete dissolution, keeping a certain rotating speed at 50 ℃ for reaction for 3 hours after finishing dripping, finally adding 100mL of PBS solution for dilution, putting the diluted solution into a dialysis bag with the molecular weight cutoff of 8000-plus 12000D, dialyzing the solution for 7 days at 60 ℃ by using deionized water, replacing the deionized water for 5 times every day, and freeze-drying the obtained gelatin solution after dialysis is finished to finally obtain the methacrylic anhydride modified gelatin.
Preferably, the adding mass ratio of the methacrylic anhydride modified gelatin and the sodium alginate in the step 1) is 65-75: 5-15, and the total mass concentration of the methacrylic anhydride modified gelatin and the sodium alginate in the mixed solution A is 6-8%.
Preferably, the total mass percentage concentration of the methacrylic anhydride modified gelatin and the sodium alginate in the mixed solution A is 8%.
Preferably, the adding ratio of the tetramethylethylenediamine in the step 2) to the mixed solution A is 2.5ml to 80 mg.
Preferably, the solvent of the ammonium persulfate solution in the step 2) is deionized water, the mass percentage concentration of the deionized water is 1%, and the ammonium persulfate solution is stored at the temperature of-4 ℃.
Preferably, the adding ratio of the ammonium persulfate solution in the step 2) to the mixed solution A is 50ml to 80 mg.
Preferably, the mold in the step 3) is a cylindrical silica gel mold with the diameter of 7.8mm and the height of 5.3 mm.
2. The injectable gelatin-sodium alginate porous cryogel with shape memory obtained by the preparation method is in a loose and porous cellular structure in a microscopic mode.
Further, the penetration rate of the porous cryogel pores is 17% -43%, and the swelling rate in 2 hours is 7.4-10.6.
The invention has the beneficial effects that: the invention takes natural gelatin and sodium alginate with good biocompatibility as raw materials, adopts low-temperature freezing polymerization to prepare a novel injectable cryogel material with a shape memory function, and overcomes the defects of poor biocompatibility, poor mechanical property and difficult realization of injectability of the conventional cryogel by simulating the structure of a natural extracellular matrix. The penetration rate of the frozen gel material prepared by the optimization method is 17-43%, and the swelling rate in 2 hours is 7.4-10.6.
Drawings
FIG. 1 shows an electron micrograph of gelatin-sodium alginate porous cryogel prepared in example 1 at 100X.
FIG. 2 shows an x 100-fold electron micrograph of the gelatin-sodium alginate porous cryogel prepared in example 2.
FIG. 3 shows an electron micrograph of gelatin-sodium alginate porous cryogel prepared in example 3 at x100 times.
FIG. 4 shows that the cryogel has an excellent shape memory function.
Figure 5 shows that the cryogel has excellent injectability.
Detailed Description
The invention will be explained in more detail below with reference to specific embodiments and the accompanying drawings.
The methacrylic anhydride modified gelatin used in the following examples was prepared as follows: weighing 5g of A type gelatin, dissolving the gelatin in 50mL PBS, heating the gelatin by using a magnetic stirrer, stirring the gelatin at a constant speed, and slowly dripping 5mL of methacrylic anhydride solution after the gelatin is completely dissolved. And after the dropwise addition is finished, keeping a certain rotating speed and reacting for 3 hours at 50 ℃, finally adding 100mL of PBS (phosphate buffer solution) for dilution, putting the diluted solution into a dialysis bag with the molecular weight cutoff of 8000-12000D, dialyzing for 7 days at 60 ℃ by using deionized water, replacing the deionized water for 5 times every day, and after the dialysis is finished, carrying out freeze drying treatment on the obtained gelatin solution to finally obtain the methacrylic anhydride modified gelatin.
Example 1
The preparation process of injectable porous gel of gelatin-sodium alginate with shape memory includes the following steps:
1) weighing 70mg of methacrylic anhydride modified gelatin and 5mg of sodium alginate, dissolving in 1ml of PBS solution, and putting into an oven at 25-60 ℃ to completely dissolve;
2) adding 2.5ml of tetramethylethylenediamine into the solution obtained in the step 1);
3) weighing 0.8g of Ammonium Persulfate (APS) to dissolve in 8ml of PBS to form an APS solution with the mass percentage concentration of 10%;
4) adding 50 mu l of APS solution obtained in the step 3) into the mixture obtained in the step 2), and uniformly mixing;
5) sucking the mixture obtained in the step 4) into a cylindrical silica gel mold with the diameter of 7.8mm and the height of 5.3mm in time, and freezing at-20 ℃ for 18 hours to prepare the gelatin-sodium alginate porous cryogel.
The hydrogel material obtained in example 1 was subjected to electron microscope irradiation to obtain an x50 electron microscope image as shown in fig. 1, and fig. 1 shows that the hydrogel material prepared in this example microscopically has a loose porous honeycomb structure and has a high porosity.
The physical and chemical properties of the material prepared in the embodiment are further tested, the pore penetration rate of the prepared hydrogel material is 23% -27%, and the swelling rate in 2 hours is 7.5-7.8.
Example 2
The preparation process of injectable porous gel of gelatin-sodium alginate with shape memory includes the following steps:
1) weighing 70mg of methacrylic anhydride modified gelatin and 10mg of sodium alginate, dissolving in 1ml of PBS solution, and putting into an oven at 25-60 ℃ to completely dissolve;
2) adding 2.5ml of Tetramethylethylenediamine (TEMED) into the solution obtained in the step 1);
3) weighing 0.8g of Ammonium Persulfate (APS) to dissolve in 8ml of PBS to form an APS solution with the mass concentration of 10%;
4) adding 50 mu l of APS solution obtained in the step 3) into the mixture obtained in the step 2), and uniformly mixing;
5) sucking the mixture obtained in the step 4) into a cylindrical silica gel mold with the diameter of 7.8mm and the height of 5.3mm in time, and freezing at-20 ℃ for 18 hours to prepare the gelatin-sodium alginate porous cryogel.
Example 3
1) Weighing 65mg of methacrylic anhydride modified gelatin and 15mg of sodium alginate, dissolving in 1ml of PBS solution, and putting into an oven at 25-60 ℃ to completely dissolve;
2) adding 2.5ml of Tetramethylethylenediamine (TEMED) into the solution obtained in the step 1);
3) weighing 0.8g of Ammonium Persulfate (APS) to dissolve in 8ml of PBS to form an APS solution with the mass concentration of 10%;
4) adding 50 mu l of APS solution obtained in the step 3) into the mixture obtained in the step 2), and uniformly mixing;
5) sucking the mixture obtained in the step 4) into a cylindrical silica gel mold with the diameter of 7.8mm and the height of 5.3mm in time, and freezing at-20 ℃ for 18 hours to prepare the gelatin-sodium alginate porous cryogel.
The samples obtained in example 2 and example 3 were tested according to the detection method described in example 1 to obtain the electron micrographs shown in fig. 2 and fig. 3, respectively, and the x50 times electron micrographs of the samples obtained in examples 2 and 3 also show a loose and porous honeycomb structure with high porosity. The hole penetration rate is 17-43%, and the swelling rate in 2 hours is 7.4-10.6.
Comparative examples
Preparation of a hydrogel comprising the steps of:
1) weighing 80mg of methacrylic anhydride modified gelatin, dissolving in 1ml of PBS solution, and putting into a baking oven at 25-60 ℃ to completely dissolve the gelatin;
2) adding 2.5ml of Tetramethylethylenediamine (TEMED) into the solution obtained in the step 1);
3) weighing 0.8g of Ammonium Persulfate (APS) to dissolve in 8ml of PBS to form an APS solution with the mass concentration of 10%;
4) adding 50 mu l of APS solution obtained in the step 3) into the mixture obtained in the step 2), and uniformly mixing;
5) sucking the mixture obtained in the step 4) into a cylindrical silica gel mold with the diameter of 7.8mm and the height of 5.3mm in time, and freezing at-20 ℃ for 18 hours to prepare the gelatin-sodium alginate porous cryogel.
The samples obtained in the comparative examples were tested in the test manner described in example 1 and had a pore penetration in the range of 16% to 18% and a swelling ratio (2h) in the range of 7.3 to 7.6.
The embodiments can demonstrate that the cryogel prepared from methacrylic anhydride modified gelatin and sodium alginate has an excellent through-hole structure, optimizes the defects of poor mechanical property and swelling property of common cryogels, simulates the composition and structure of natural extracellular matrix, and solves the problem of poor biocompatibility of the existing injectable tissue engineering scaffold material.
Further, the shape memory function test of the cryogel prepared in example 1 is performed, and fig. 4 shows that when the shape of the cryogel is changed after being compressed by an external force, and simultaneously, the internal moisture is continuously squeezed out, and when the external force is removed, the cryogel can rapidly absorb the surrounding water and rapidly recover to the original shape (as shown in fig. 4A-4C), and meanwhile, the cryogel material can also rapidly recover to the original shape (as shown in fig. 4D-4F) after being bent and deformed by the external force, which can show that the cryogel has an excellent shape memory function.
The cryogels prepared in example 1 were further tested for injectability, and fig. 5 shows that the cryogels have injectability, the cryogels can be easily pushed out of the syringe, and the injected cryogels can rapidly absorb water and return to their original hydrated form (see fig. 5A-5C).
In conclusion, the invention takes natural gelatin and sodium alginate with good biocompatibility as raw materials, adopts low-temperature freezing polymerization to prepare a novel injectable cryogel material with a shape memory function, and overcomes the defects of poor biocompatibility, poor mechanical property and difficult realization of injectability of the existing cryogel by simulating the structure of a natural extracellular matrix. The penetration rate of the frozen gel material prepared by the optimization method is 17-43%, and the swelling rate in 2 hours is 7.4-10.6.
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.
Claims (6)
1. A preparation method of injectable gelatin-sodium alginate porous cryogel with shape memory is characterized by comprising the following steps:
1) mixing and dissolving methacrylic anhydride modified gelatin and sodium alginate in a PBS solution at the temperature of 25-60 ℃ in an oven, and recording the mixed solution A;
2) sequentially adding tetramethyl ethylenediamine and an ammonium persulfate solution into the mixed solution A obtained in the step 1), uniformly mixing, and recording a mixed solution B, wherein the adding ratio of the tetramethyl ethylenediamine to the mixed solution A is 2.5ml to 80mg, and the adding ratio of the ammonium persulfate solution to the mixed solution A is 50ml to 80 mg;
3) sucking the mixture B obtained in the step 2) into a mold in time, and freezing the mixture B for 18 to 24 hours at a temperature of between 20 ℃ below zero and 80 ℃ below zero to prepare gelatin-sodium alginate porous cryogel;
the preparation method of the methacrylic anhydride modified gelatin in the step 1) comprises the following steps: weighing 5g of A type gelatin, dissolving the gelatin in 50mL of PBS solution, heating the gelatin to 50 ℃ by using a magnetic stirrer, stirring the gelatin at a constant speed, slowly dripping 5mL of methacrylic anhydride solution after complete dissolution, keeping a certain rotating speed at 50 ℃ for reaction for 3 hours after finishing dripping, finally adding 100mL of PBS solution for dilution, putting the diluted solution into a dialysis bag with the molecular weight cutoff of 8000-plus 12000D, dialyzing the solution by using deionized water at 60 ℃ for 7 days, replacing the deionized water for 5 times every day, and freeze-drying the obtained gelatin solution after dialysis is finished to finally obtain the methacrylic anhydride modified gelatin.
2. The preparation method of the injectable gelatin-sodium alginate porous cryogel with shape memory according to claim 1, wherein the addition mass ratio of the methacrylic anhydride modified gelatin to the sodium alginate in the step 1) is 65-75: 5-15, and the total mass percentage concentration of the methacrylic anhydride modified gelatin and the sodium alginate in the mixed solution A is 6-8%.
3. The method for preparing the injectable gelatin-sodium alginate porous cryogel with shape memory as claimed in claim 2, wherein the total concentration of methacrylic anhydride modified gelatin and sodium alginate in the mixed solution A is 8%.
4. The preparation method of the injectable gelatin-sodium alginate porous cryogel with shape memory according to claim 1, wherein the solvent of the ammonium persulfate solution in the step 2) is deionized water with a mass percentage concentration of 1%, and the ammonium persulfate solution is stored at a temperature of-4 ℃.
5. The method for preparing the injectable gelatin-sodium alginate porous cryogel with shape memory according to claim 1, wherein the mold of step 3) is a cylindrical silica gel mold with a diameter of 7.8mm and a height of 5.3 mm.
6. The injectable gelatin-sodium alginate porous cryogel with shape memory of claim 5, wherein the porous cryogel has a pore penetration rate of 17% to 43% and a swelling rate of 7.4 to 10.6 at 2 hours.
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CN114405486B (en) * | 2021-11-17 | 2023-06-23 | 重庆科技学院 | Preparation method of porous cryogel with antifouling and antibacterial functions and dye adsorption preventing functions |
CN114196058B (en) * | 2021-12-17 | 2023-04-18 | 浙江工业大学 | Amphoteric ion gel sponge and preparation method thereof |
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