CN111330089B - Preparation method of fully-degradable magnetic repair hydrogel for bone repair - Google Patents
Preparation method of fully-degradable magnetic repair hydrogel for bone repair Download PDFInfo
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Abstract
The invention discloses a preparation method of fully-degradable magnetic repair hydrogel for bone repair, which comprises the following steps: step 1: preparing a mixed aqueous solution containing iron ions, calcium ions and dopamine; step 2: stirring uniformly under the atmosphere protection condition, dripping a phosphate solution into the mixture, and fully reacting to obtain dopamine dispersed nano magnetic hydroxyapatite particles; and step 3: dissolving gelatin in phosphate buffer, adding methacrylic anhydride after full dissolution, dialyzing and freeze-drying after full reaction to obtain double-bonded gelatin; and 4, step 4: adding nano magnetic hydroxyapatite particles into a double-bonded gelatin aqueous solution, and adding sodium persulfate to form a hydrogel pre-polymerization solution; applying magnetic fields on two sides of the hydrogel prepolymerization solution to polymerize the hydrogel to form the required hydrogel; the preparation method is simple and easy to operate, and the obtained nano particles have controllable particle size and can form directional arrangement in the hydrogel under the action of a magnetic field.
Description
Technical Field
The invention relates to the technical field of biological materials, in particular to a preparation method of a fully-degradable magnetic repair hydrogel for bone repair.
Background
The proportion of bone defects of a human body is increased year by year due to factors such as trauma, infection, tumor excision and the like, and the performance requirements and the requirements of bone repair materials are greatly increased. In recent years, hydrogel is developed and used as a tissue repair material due to its high water content, the advantages of extracellular matrix imitation and the like, and particularly, introduction of a nanomaterial with osteoinductivity into hydrogel can remarkably promote osteogenic repair. Among the various nano materials, nano hydroxyapatite is used as a main inorganic salt component in natural bones and can be integrated with host bones under physiological conditions and release ions so as to promote the expression of osteogenesis related genes. Based on the bionic principle, the ideal bone repair hydrogel not only needs components similar to bones, but also has an extremely important oriented structure of nano-hydroxyapatite. The method for effectively forming the anisotropic hydroxyapatite is to assemble the magnetic ferroferric oxide on the surface of the nano hydroxyapatite and drive the orientation of the magnetic hydroxyapatite by utilizing a magnetic field. However, both the nano hydroxyapatite and the nano ferroferric oxide have high surface energy and are easy to agglomerate, and the low affinity between the hydroxyapatite and the ferroferric oxide limits the final generation of the magnetic hydroxyapatite. In addition, the hydrogel matrix should also have excellent mechanical properties, biocompatibility and degradability to meet the requirements of implantation. Therefore, it remains a challenge to develop a fully degradable magnetic hydrogel that is osteoinductive.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a preparation method of fully-degradable magnetic repair hydrogel which is simple in method, easy to operate, controllable in nanoparticle size and capable of forming directional arrangement in hydrogel under the action of a magnetic field and used for bone repair.
The technical scheme adopted by the invention is as follows:
a preparation method of fully-degradable magnetic repair hydrogel for bone repair comprises the following steps:
step 1: preparing mixed aqueous solution containing iron ions, calcium ions and dopamine, and adding Fe2 in the mixed aqueous solution+：Fe3+The molar ratio of (A) to (B) is 0.5 to 1; dopamine and Fe2+The mass ratio of (A) to (B) is 0.01-1; ca2+：Fe2+The molar ratio of (A) to (B) is 0-4;
step 2: stirring uniformly under the atmosphere protection condition, dripping a phosphate solution into the mixture, and fully reacting to obtain dopamine dispersed nano magnetic hydroxyapatite particles; wherein Ca2+And PO4 3-The molar ratio of (A) to (B) is 1.5-2;
and step 3: dissolving gelatin in phosphate buffer, adding methacrylic anhydride after full dissolution, dialyzing and freeze-drying after full reaction to obtain double-bonded gelatin; wherein the addition amount of the methacrylic anhydride is 0.4-0.8 mL per 1g of gelatin;
and 4, step 4: adding the nano magnetic hydroxyapatite particles obtained in the step 2 into the double-bonded gelatin aqueous solution obtained in the step 3, and adding sodium persulfate to form hydrogel pre-polymerization liquid; applying magnetic fields in parallel at two sides of the hydrogel prepolymerization solution, wherein the magnetic field intensity is 10-50 mt, and the hydrogel can be polymerized to form the required hydrogel; wherein the mass ratio of the nano magnetic hydroxyapatite particles to the double-bonded gelatin is 5-20%: 1.
further, during the step 2 reaction, the pH value of the solution is maintained to be more than 10.
Further, the reaction in the step 2 is carried out at 80 ℃, and the reaction time is 60-120 min.
Further, in the step 2, the pH value of the solution is maintained by dropwise adding sodium hydroxide or ammonia water.
Further, the phosphate solution was added dropwise at a rate of 2 drops per second.
Further, in the step 3, in the process of dissolving gelatin in the phosphate buffer, stirring for 30min at the temperature of 60 ℃ in a water bath to dissolve the gelatin; adding methacrylic anhydride, and stirring for 30 hours under the condition of water bath at 50 ℃ for reaction.
Further, the mass ratio of the sodium persulfate to the double-bonded gelatin in the step 4 is 1: 10.
The invention has the beneficial effects that:
(1) the preparation method is simple and easy to operate, the particle size of the obtained nano particles is controllable, and the nano particles can form directional arrangement in the hydrogel under the action of a magnetic field;
(2) the modified natural polymer is used as the hydrogel matrix material, and has good mechanical properties, biocompatibility, mechanical properties and complete degradability.
Drawings
FIG. 1 is an XRD pattern of the magnetic nano-hydroxyapatite prepared by the invention.
Fig. 2 is a schematic diagram of a magnetic hysteresis loop of the magnetic nano-hydroxyapatite prepared by the invention.
Fig. 3 is an SEM image of the magnetic nano-hydroxyapatite prepared by the present invention.
FIG. 4 is an SEM image of the directionally aligned magnetic hydroxyapatite-loaded natural polymer hydrogel prepared by the present invention.
Detailed Description
The invention is further described with reference to the following figures and specific embodiments.
A preparation method of fully-degradable magnetic repair hydrogel for bone repair comprises the following steps:
step 1: preparing mixed aqueous solution containing iron ions, calcium ions and dopamine, and adding Fe2 in the mixed aqueous solution+：Fe3+The molar ratio of (A) to (B) is 0.5 to 1; dopamine and Fe2+The mass ratio of (A) to (B) is 0.01-1; ca2+：Fe2+The molar ratio of (a) to (b) is 0-4, wherein the iron ions are FeCl2·4H2O and FeCl3·6H 20 calcium ion is Ca (NO)3)2·4H2And O.
Step 2: stirring evenly under the atmosphere protection condition, dripping phosphate solution into the mixture, and fully reacting to obtain the polysaccharideA bamine dispersed nano-magnetic hydroxyapatite particle; wherein Ca2+And PO4 3-The molar ratio of (A) to (B) is 1.5-2; wherein the atmosphere is nitrogen, the pH value of the solution is maintained to be more than 10 in the reaction process, and the pH value of the solution is maintained by dropwise adding sodium hydroxide or ammonia water. The reaction is carried out at 80 ℃ for 60-120 min.
And step 3: dissolving gelatin in phosphate buffer, adding methacrylic anhydride after full dissolution, dialyzing and freeze-drying after full reaction to obtain double-bonded gelatin; wherein the addition amount of the methacrylic anhydride is 0.4-0.8 mL per 1g of gelatin;
and 4, step 4: adding the nano magnetic hydroxyapatite particles obtained in the step 2 into the double-bonded gelatin aqueous solution obtained in the step 3, and adding sodium persulfate to form hydrogel pre-polymerization liquid; applying magnetic fields in parallel at two sides of the hydrogel prepolymerization solution, wherein the magnetic field intensity is 10-50 mt, and the hydrogel can be polymerized to form the required hydrogel; wherein the mass ratio of the nano magnetic hydroxyapatite particles to the double-bonded gelatin is 5-20%: 1. dissolving gelatin in phosphate buffer solution, stirring at 60 deg.C in water bath for 30 min; adding methacrylic anhydride, and stirring for 30 hours under the condition of water bath at 50 ℃ for reaction.
Example 1
A preparation method of fully-degradable magnetic repair hydrogel for bone repair comprises the following steps:
step 1: under the condition of room temperature, 8 g of FeCl2·4H2O，20.8 g FeCl3·6H 20, 0.1 g dopamine was put into a 500 mL three-necked flask, to which 25 mL H was added2O。
Step 2: and introducing nitrogen into the solution to remove oxygen in the beaker, ensuring that the whole system is in an oxygen-free environment, stirring for 30min at 1000 r/min by using a mechanical stirrer, and preparing 250 mL of aqueous solution containing 15 g of NaOH. Stirring for 30min, heating the three-neck flask to 80 ℃ through a water bath kettle, and dropwise adding a sodium hydroxide solution into the three-neck flask at the speed of 2 drops per second; after the dropwise addition is finished, stirring is continuously carried out for 30min at the temperature of 80 ℃; generating the magnetic nano-hydroxyapatite chelated with the dopamine. After the reaction is finished, separating the nano particles by a magnetic separation method, and washing the nano particles with 100 mL of deoxygenated deionized water for three times each time to obtain the dopamine chelated magnetic nano hydroxyapatite particles.
And step 3: 5 g of gelatin was dissolved in 50 mL of phosphate buffer (pH = 7.4), and the gelatin was dissolved in a 60 ℃ water bath with stirring for 30min, followed by slow addition of 4 mL of methacrylic anhydride at 0.5 mL/min. The mixed solution was stirred continuously in a 50 ℃ water bath for 3 hours. After the reaction, the solution was filled into a dialysis bag (MW = 14000) and dialyzed at 40 ℃ for 7 days. And (4) freeze-drying the dialyzed liquid to obtain the double-bonded gelatin.
And 4, step 4: and (3) dissolving 1g of double-bonded gelatin prepared in the step (3) in 10 mL of water, adding 0.1 g of magnetic nano-hydroxyapatite obtained in the step (1) into the solution, adding 0.1 g of sodium persulfate, applying a magnetic field to two sides of the hydrogel prepolymerization solution, and polymerizing to form the required hydrogel.
Fig. 1a is an XRD pattern of the magnetic nano-hydroxyapatite particles obtained in step 2 of this example. Fig. 2a is a magnetic hysteresis chart of the magnetic nano-hydroxyapatite particles obtained in step 2 of this example. Fig. 3a is an SEM image of the magnetic nano-hydroxyapatite particles obtained in step 2 of this example. Fig. 4a is a diagram of the directional arrangement of the magnetic nano-hydroxyapatite particles obtained in step 4 of this embodiment in the hydrogel.
Example 2
A preparation method of fully-degradable magnetic repair hydrogel for bone repair comprises the following steps:
step 1: under the condition of room temperature, 6 g of FeCl2·4H2O，15.6 g FeCl3·6H 20, 0.1 g dopamine and 2.972 g Ca (NO)3)2·4H2O was charged into a 500 mL three-necked flask, and 25 mL of H was added thereto2O。
Step 2: introducing nitrogen into the solution to remove oxygen in the beaker, ensuring that the whole system is in an oxygen-free environment, stirring for 30min at 1000 r/min by using a mechanical stirrer, and simultaneously preparing 250 mL of aqueous solution containing 15 g of NaOH and 0.99 g of (NH)4)2HPO4An aqueous solution of (a). Stirring for 30min, heating to 80 deg.C in water bath, and adding sodium hydroxide solutionAnd the phosphoric acid diamine solution is added into the three-neck flask drop by drop at the speed of 2 drops per second; after the dropwise addition is finished, stirring is continuously carried out for 30min at the temperature of 80 ℃; generating the magnetic nano-hydroxyapatite chelated with the dopamine. After the reaction is finished, separating the nano particles by a magnetic separation method, and washing the nano particles with 100 mL of deoxygenated deionized water for three times each time to obtain the dopamine chelated magnetic nano hydroxyapatite particles.
And step 3: 5 g of gelatin was dissolved in 50 mL of phosphate buffer (pH = 7.4), and the gelatin was dissolved in a 60 ℃ water bath with stirring for 30min, followed by slow addition of 4 mL of methacrylic anhydride at 0.5 mL/min. The mixed solution was stirred continuously in a 50 ℃ water bath for 3 hours. After the reaction, the solution was filled into a dialysis bag (MW = 14000) and dialyzed at 40 ℃ for 7 days. And (4) freeze-drying the dialyzed liquid to obtain the double-bonded gelatin.
And 4, step 4: and (3) dissolving 1g of double-bonded gelatin prepared in the step (3) in 10 mL of water, adding 0.1 g of magnetic nano-hydroxyapatite obtained in the step (1) into the solution, adding 0.1 g of sodium persulfate, applying a magnetic field to two sides of the hydrogel prepolymerization solution, and polymerizing to form the required hydrogel.
Fig. 1b is an XRD pattern of the magnetic nano-hydroxyapatite particles obtained in step 2 of this example. Fig. 2b is a magnetic hysteresis chart of the magnetic nano-hydroxyapatite particles obtained in step 2 of this embodiment. Fig. 3b is an SEM image of the magnetic nano-hydroxyapatite particles obtained in step 2 of this example. Fig. 4b is a diagram of the directional arrangement of the magnetic nano-hydroxyapatite particles obtained in step 4 of this embodiment in the hydrogel.
Example 3
A preparation method of fully-degradable magnetic repair hydrogel for bone repair comprises the following steps:
step 1: under the condition of room temperature, 4 g of FeCl2·4H2O，10.4 g FeCl3·6H 20, 0.1 g dopamine and 5.944 g Ca (NO)3)2·4H2O was charged into a 500 mL three-necked flask, and 25 mL of H was added thereto2O。
Step 2: introducing nitrogen into the solution to remove oxygen in the beaker, ensuring the whole system to be in an oxygen-free environment, and using the machineStirring with a mechanical stirrer at 1000 r/min for 30min, and simultaneously preparing 250 mL of aqueous solution containing 15 g of NaOH and 1.98 g of (NH)4)2HPO4An aqueous solution of (a). Stirring for 30min, heating the three-neck flask to 80 ℃ through a water bath kettle, and dropwise adding a sodium hydroxide solution and a phosphoric acid diamine solution into the three-neck flask at the speed of 2 drops per second; after the dropwise addition is finished, stirring is continuously carried out for 30min at the temperature of 80 ℃; generating the magnetic nano-hydroxyapatite chelated with the dopamine. After the reaction is finished, separating the nano particles by a magnetic separation method, and washing the nano particles with 100 mL of deoxygenated deionized water for three times each time to obtain the dopamine chelated magnetic nano hydroxyapatite particles.
And step 3: 5 g of gelatin was dissolved in 50 mL of phosphate buffer (pH = 7.4), and the gelatin was dissolved in a 60 ℃ water bath with stirring for 30min, followed by slow addition of 4 mL of methacrylic anhydride at 0.5 mL/min. The mixed solution was stirred continuously in a 50 ℃ water bath for 3 hours. After the reaction, the solution was filled into a dialysis bag (MW = 14000) and dialyzed at 40 ℃ for 7 days. And (4) freeze-drying the dialyzed liquid to obtain the double-bonded gelatin.
And 4, step 4: and (3) dissolving 1g of double-bonded gelatin prepared in the step (3) in 10 mL of water, adding 0.1 g of magnetic nano-hydroxyapatite obtained in the step (1) into the solution, adding 0.1 g of sodium persulfate, applying a magnetic field to two sides of the hydrogel prepolymerization solution, and polymerizing to form the required hydrogel.
Fig. 1c is an XRD pattern of the magnetic nano-hydroxyapatite particles obtained in step 2 of this example. Fig. 2c is a magnetic hysteresis chart of the magnetic nano-hydroxyapatite particles obtained in step 2 of this example. Fig. 3c is an SEM image of the magnetic nano-hydroxyapatite particles obtained in step 2 of this example. Fig. 4c is a diagram of the directional arrangement of the magnetic nano-hydroxyapatite particles obtained in step 4 of this embodiment in the hydrogel.
Example 4
A preparation method of fully-degradable magnetic repair hydrogel for bone repair comprises the following steps:
step 1: under the condition of room temperature, 2 g of FeCl2·4H2O，5.2 g FeCl3·6H 20, 0.1 g dopamine and 8.916 g Ca (NO)3)2·4H2O was charged into a 500 mL three-necked flask, and 25 mL of H was added thereto2O。
Step 2: introducing nitrogen into the solution to remove oxygen in the beaker, ensuring that the whole system is in an oxygen-free environment, stirring for 30min at 1000 r/min by using a mechanical stirrer, and simultaneously preparing 250 mL of aqueous solution containing 15 g of NaOH and (NH) containing 2.97 g of NaOH4)2HPO4An aqueous solution of (a). Stirring for 30min, heating the three-neck flask to 80 ℃ through a water bath kettle, and dropwise adding a sodium hydroxide solution and a phosphoric acid diamine solution into the three-neck flask at the speed of 2 drops per second; after the dropwise addition is finished, stirring is continuously carried out for 30min at the temperature of 80 ℃; generating the magnetic nano-hydroxyapatite chelated with the dopamine. After the reaction is finished, separating the nano particles by a magnetic separation method, and washing the nano particles with 100 mL of deoxygenated deionized water for three times each time to obtain the dopamine chelated magnetic nano hydroxyapatite particles.
And step 3: 5 g of gelatin was dissolved in 50 mL of phosphate buffer (pH = 7.4), and the gelatin was dissolved in a 60 ℃ water bath with stirring for 30min, followed by slow addition of 4 mL of methacrylic anhydride at 0.5 mL/min. The mixed solution was stirred continuously in a 50 ℃ water bath for 3 hours. After the reaction, the solution was filled into a dialysis bag (MW = 14000) and dialyzed at 40 ℃ for 7 days. And (4) freeze-drying the dialyzed liquid to obtain the double-bonded gelatin.
And 4, step 4: and (3) dissolving 1g of double-bonded gelatin prepared in the step (3) in 10 mL of water, adding 0.1 g of magnetic nano-hydroxyapatite obtained in the step (1) into the solution, adding 0.1 g of sodium persulfate, applying a magnetic field to two sides of the hydrogel prepolymerization solution, and polymerizing to form the required hydrogel.
Fig. 1d is an XRD pattern of the magnetic nano-hydroxyapatite particles obtained in step 2 of this example. Fig. 2d is a magnetic hysteresis chart of the magnetic nano-hydroxyapatite particles obtained in step 2 of this embodiment. Fig. 3d is an SEM image of the magnetic nano-hydroxyapatite particles obtained in step 2 of this example. Fig. 4d is a diagram of the directional arrangement of the magnetic nano-hydroxyapatite particles obtained in step 4 of this embodiment in the hydrogel.
From fig. 1, it can be seen that characteristic peaks of ferroferric oxide and hydroxyapatite appear in the XRD chart, which indicates that the obtained nanoparticles are magnetic hydroxyapatite. The nanoparticle of fig. 1a was prepared without the characteristic peaks of calcium and phosphate ions and only ferroferric oxide. Figures 1b, c and d the features of hydroxyapatite enhanced with increasing mass input of calcium ions and phosphate ions.
As can be seen from the hysteresis loop of fig. 2, the nanoparticles of examples 1 to 4 all have magnetic properties, and the magnetic properties decrease as the input mass of calcium ions and phosphate ions increases.
As can be seen from FIG. 3, Fe3O4The SEM photograph of (A) is spherical, the SEM photograph of hyaluronic acid HA is rod-shaped, and the rod-shaped proportion in the SEM photograph increases as the input mass of calcium ions and phosphate ions increases.
As can be seen from fig. 4, the nanoparticles obtained in examples 1 to 4 are magnetic, and therefore are aligned in the hydrogel under the action of an external magnetic field.
The hydrogel prepared by the invention has higher stability and biocompatibility in vivo, and can realize low swelling and full degradation; the magnetic nano-hydroxyapatite is used as the nano-reinforcing phase in the hydrogel, so that the mechanical property of the hydrogel can be improved, and meanwhile, the hydroxyapatite in the nano-particles is the main inorganic component of the hard tissue of an organism, so that the bone repair is facilitated. The structure of the bionic bone of the magnetic hydroxyapatite in the hydrogel is realized by utilizing an external magnetic field. The magnetic ferroferric oxide influences the adhesion, proliferation, migration and differentiation of various cells under the action of a magnetic field, promotes osteogenic differentiation of the cells and is beneficial to bone repair. The magnetic ferroferric oxide has high stability in vivo and can be stored in vivo for a long time and finally discharged through the kidney. The magnetic field HAs a promoting effect on bone repair, HA is an inorganic component required by bone repair, and the synergistic effect of the HA and the HA further improves the bone repair effect. Dopamine can realize good dispersibility and chelation on the nanoparticles, and compared with the traditional preparation method, the dopamine can enable the size of the nanoparticles to be smaller, thereby being beneficial to in vivo renal excretion and further research.
Claims (6)
1. A preparation method of fully-degradable magnetic repair hydrogel for bone repair is characterized by comprising the following steps:
step 1: preparing mixed aqueous solution containing iron ions, calcium ions and dopamine, and adding Fe2 in the mixed aqueous solution+：Fe3+The molar ratio of (A) to (B) is 0.5 to 1; dopamine and Fe2+The mass ratio of (A) to (B) is 0.01-1; ca2+：Fe2+The molar ratio of (A) to (B) is 0-4;
step 2: stirring uniformly under the atmosphere protection condition, dripping a phosphate solution into the mixture, and fully reacting to obtain dopamine dispersed nano magnetic hydroxyapatite particles; wherein Ca2+And PO4 3-The molar ratio of (A) to (B) is 1.5-2;
and step 3: dissolving gelatin in phosphate buffer, adding methacrylic anhydride after full dissolution, dialyzing and freeze-drying after full reaction to obtain double-bonded gelatin; wherein the addition amount of the methacrylic anhydride is 0.4-0.8 mL per 1g of gelatin;
and 4, step 4: adding the nano magnetic hydroxyapatite particles obtained in the step 2 into the double-bonded gelatin aqueous solution obtained in the step 3, and adding sodium persulfate to form hydrogel pre-polymerization liquid; applying magnetic fields in parallel at two sides of the hydrogel prepolymerization solution, wherein the magnetic field intensity is 10-50 mt, and the hydrogel can be polymerized to form the required hydrogel; wherein the mass ratio of the nano magnetic hydroxyapatite particles to the double-bonded gelatin is 5-20%: 1; in the process that the gelatin is dissolved in the phosphate buffer solution in the step 3, stirring for 30min at the water bath condition of 60 ℃ for dissolving; adding methacrylic anhydride, and stirring for 30 hours under the condition of water bath at 50 ℃ for reaction.
2. The method for preparing fully degradable magnetic repair hydrogel for bone repair according to claim 1, wherein during the reaction of step 2, the pH of the solution is maintained to be greater than 10.
3. The method for preparing the fully degradable magnetic repair hydrogel for bone repair according to claim 1, wherein the reaction in the step 2 is carried out at 80 ℃ for 60-120 min.
4. The method for preparing fully degradable magnetic repair hydrogel for bone repair according to claim 2, wherein the pH value of the solution is maintained by dropping sodium hydroxide or ammonia water in step 2.
5. The method for preparing a fully degradable magnetic repair hydrogel for bone repair according to claim 1 wherein the phosphate solution is added dropwise at a rate of 2 drops per second.
6. The method for preparing fully degradable magnetic repair hydrogel for bone repair according to claim 1, wherein the mass ratio of sodium persulfate to doubly bonded gelatin in step 4 is 1: 10.
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