CN113004551B - Construction method of metal ion composite crosslinking system of alginate bone tissue engineering scaffold - Google Patents
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Abstract
The construction method of the alginate bone tissue engineering scaffold metal ion composite crosslinking system comprises the steps of purifying an alginate material, preparing an alginate precursor solution, and preparing a multi-ion composite crosslinking agent: weighing anhydrous CaCl 2 0.4g，SrCl 2 0.4g，ZnCl 2 0.16g and Mg Cl 2 0.244g, dissolved in 100mL double distilled water to prepare a mixed solution; alginate gel synthesis: heating the precursor solution to 60 ℃, magnetically stirring, and placing in a 37 ℃ water bath incubator; and respectively taking the precursor solution and the compound cross-linking agent with equal volumes, fully and uniformly mixing the precursor solution and the compound cross-linking agent by using a sterile medical three-way pipe, injecting the mixture into any designated position within 10 seconds, and forming solid gel after 1 minute. The composite crosslinking system of the invention ensures the crosslinking concentration of the alginate gel, and ensures the biological safety of the alginate gel in the process of slowly releasing crosslinking ions by controlling the respective concentrations of four ions in the crosslinking agent.
Description
Technical Field
The invention belongs to the field of hydrogel material crosslinking methods, and particularly relates to a method for constructing an alginate bone tissue engineering scaffold metal ion composite crosslinking system.
Background
In recent years, with the rise of minimally invasive concepts, injectable bone tissue engineering has attracted a great deal of attention. Under the repair mode, the seed cells and the hydrogel bracket enter the bone defect part in an injection mode, so that the treatment has the advantages of small wound, simple operation and capability of filling any defect shape. Alginate is a high molecular compound extracted from brown algae, is formed by connecting beta-D-mannuronic acid (M unit) and alpha-L-guluronic acid (G unit) by 1, 4-glycosidic bond, and is the only natural polysaccharide which can be dissolved in water at room temperature to form sol. Alginate has good biocompatibility and rich sources, and has been approved by the U.S. FDA for pharmaceutical dosage form development and food industry. The G unit in the alginate can be chelated with divalent cations to form an eggshell-like internal structure, and then a solid hydrogel is formed, the structure of the solid hydrogel is similar to the microenvironment of cell growth, and the solid hydrogel has good prospect in injectable bone tissue engineering. However, as a scaffold material, alginate hydrogels still have the disadvantages: (1) divalent cations in the gel are easy to exchange with the surrounding environment and lose the gel property; (2) the gel does not have osteoinductive effect. Accordingly, efforts to overcome the above-mentioned drawbacks have become critical for the application of alginate hydrogel scaffold materials.
Alginate is usually ion crosslinked with divalent cations, but divalent cations in the gel are easily ion exchanged with the surrounding environment, which in turn leads to dissolution of the gel. At present, a certain concentration of calcium ions are conventionally used as crosslinking ions for the alginate crosslinking agent at home and abroad, but the calcium ions are very easy to replace monovalent cations in physiological environments, so that gel collapse is caused. In the applicant's early studies, using strontium ions as a cross-linking agent for alginate gels, it was found that an equal concentration of calcium/strontium ion mix cross-linking was effective in improving the stability of the gel and promoting the adhesion and proliferation of osteoblasts to the gel (Zhang Man, li, xu Ting, liu Jie, et al, influence of strontium-containing cross-linked sodium alginate gels on the adhesion and proliferation of preosteoblasts [ J ], open sea oral medicine, 2019,28 (2): 123-127 ]) but this cross-linking agent still had a number of problems. The addition of strontium ions is beneficial to the improvement of gel stability and mechanical strength, but too high strontium ions have toxicity to cells, and cannot be excessively introduced as a crosslinking agent. Thus, the solution of calcium/strontium ions as a cross-linking agent still has many limitations.
Disclosure of Invention
The invention aims to provide a method for constructing a metal ion composite crosslinking system of an alginate bone tissue engineering scaffold, which utilizes four metal ions to construct a crosslinking system of alginate gel, thereby improving the physical and chemical properties, the structural stability and the biological properties related to bone formation of the gel.
Alginate is usually cross-linked by calcium ions, and alpha-L-guluronic acid (G units) in alginate reacts with calcium ions in a chelate reaction to form an "eggshell-like" structure, thereby changing the liquid gel precursor solution into a solid gel with certain strength. However, after the gel is formed, calcium ions in the gel are easily replaced and released with monovalent cations in a physiological environment, so that the internal structure of the gel is collapsed and decomposed, and the gel material is used as a tissue engineering scaffold to fail. Studies have shown that in addition to calcium ions, strontium ions, zinc ions, magnesium ions can also undergo chelation reactions with alginate G units, which in turn form solid gels. Research has found that, on the one hand, these ions form gels with better stability than calcium ions; on the other hand, these ions at appropriate concentrations will be beneficial in increasing osteoblast activity, promoting healing of bone defects, such as released Sr 2+ Can stimulate new bone formation and inhibit osteoclast activity, while Zn 2+ Can increase alkaline phosphatase activity, thereby increasing osteoblast activity. In view of the natural slow release characteristics of the alginate gel materials, the addition and slow release of the ions can effectively improve the structural stability and the osteogenic bioactivity of the alginate. However, both zinc ions and magnesium ions belong to trace elements of human bodies, and researches show that too high concentration of both zinc ions and magnesium ions can cause toxicity to human bodies, and too low concentration of both zinc ions and magnesium ions can not crosslink alginate gel to form solid gel, so that the two ions are rarely used as crosslinking agents of biomedical alginate gel.
In response to this problem, the applicant has made the following thinking, since Sr 2+ 、Zn 2+ ，Mg 2+ Has the effect of having an excessively high concentration when in useToxicity, then what is its toxicity? What will the toxicity be? What is the toxicity change when the ratio of the added components is different? In this way, the applicant has tried to use Ca while performing reverse thinking 2+ 、Sr 2+ 、Zn 2+ ，Mg 2+ The four divalent cations together complete the ionic crosslinking process and the crosslinking effect at different concentrations was observed. As a result, it was found that the simultaneous use of four divalent cations does not mean monotonic increase in toxicity, but that the combination of four divalent cations in different concentration ratios in the reaction system produces different effects. After a large number of repeated experiments, it was found that the toxicity of the cross-linking system obtained by the four particles in different proportions is different, and more importantly, after the proportion is properly adjusted, an optimal adding proportion can be obtained by analysis on the premise of ensuring the cross-linking effect, so that the optimal compound concentration proportion of the four ions serving as alginate injectable bone tissue engineering scaffolds can be finally screened out, as shown in table 1.
TABLE 1 alginate Metal ion Complex Cross-linking agent ion concentrations
The construction method of the metal ion composite crosslinking system of the alginate bone tissue engineering scaffold is characterized by comprising the following steps:
(1) Purification of alginate material: alginate with average molecular weight 4000 was added to triple distilled deionized water to prepare a solution with a concentration of 8% wt; placing a regenerated cellulose dialysis bag with the molecular weight cut-off of 3500 in 2% W/V sodium bicarbonate and 1mM EDTA with the pH of 8.0, boiling for 10 minutes, and thoroughly cleaning with distilled water; adding the alginate solution into a treated dialysis bag, sealing two ends of the dialysis bag, and placing the dialysis bag into a beaker; adding deionized water to submerge the dialysis bag, placing on a magnetic stirrer to stir for 72 hours, replacing the deionized water every 4 hours in the first 12 hours, and then replacing every 12 hours; after dialysis, collecting the solution in the dialysis bag, and freeze-drying for 24 hours to obtain alginate powder;
(2) Preparation of alginate precursor solution: weighing 4g of alginate powder and adding the alginate powder into a reagent bottle containing 100mL of 0.01M PBS solution, and placing the reagent bottle on a magnetic stirrer to stir for 12 hours so as to completely dissolve the alginate powder; titrating the alginate precursor solution to a pH of 7.4 using 0.1M dilute hydrochloric acid or sodium hydroxide solution, preparing an alginate solution having a final concentration of 4% wt, and filter-sterilizing the alginate solution using a 0.22 μm sterile filter;
(3) Configuration of the polyion composite crosslinking agent: weighing anhydrous CaCl 2 0.4g，SrCl 2 0.4g，ZnCl 2 0.116g and Mg Cl 2 0.244g, dissolving in 100mL double distilled water to prepare a mixed solution, enabling the concentration of the mixed solution to be 2 times of the final concentration of the cross-linking agent to be prepared, sterilizing the mixed solution by using a 0.22 mu m sterile filter, storing in a sterile reagent bottle, and uniformly mixing by spiral oscillation before use;
(4) Alginate gel synthesis: heating the alginate solution obtained in the step 2 to 60 ℃ before each use, magnetically stirring for 1h, and placing in a 37 ℃ water bath constant temperature box; and (3) respectively taking the alginate precursor solution with equal volume and the composite cross-linking agent obtained in the step (3), fully and uniformly mixing the alginate precursor solution and the composite cross-linking agent by using a sterile medical three-way pipe, injecting the mixture into any designated position within 10 seconds, and forming solid gel after 1 minute.
In view of the popularity of sodium alginate, the alginate is preferably sodium alginate.
Under the compound concentration proportion, the crosslinking reaction effect is mainly characterized by the following:
(1) The composite crosslinking system ensures the crosslinking concentration of the alginate gel, and can ensure the formation of the alginate gel and the stability of more excellent materials in physiological environment. Compared with the traditional simple calcium ion crosslinked alginate gel, the gel structure of the invention is more uniform and stable (see figure 1), and the gel strength is obviously enhanced (see figure 2).
(2) By utilizing the characteristic of ion release after alginic acid crosslinking, a local microenvironment favorable for bone growth is formed, and the osteogenic activity of the alginate gel is effectively improved. Compared with the traditional simple calcium ion crosslinked alginate gel, the novel gel can continuously promote the alkaline phosphatase activity of osteoblasts at different time points, which proves that the novel gel has better osteogenic activity (see figure 6)
(3) The biological safety of the alginate gel in the process of slowly releasing the crosslinking ions is ensured by controlling the respective concentrations of the four ions in the crosslinking agent. The observation by using a scanning electron microscope shows that compared with the traditional calcium ion-only crosslinked alginate gel, the novel gel surface has more cell adhesion, and the cell adhesion state is better than that of the traditional gel surface (see figures 3 and 4), so that the novel gel has better biosafety.
(4) On the premise of unchanged concentration of the calcium and strontium ions, the zinc ions and the magnesium ions have forward synergistic effect, and toxicity is reduced. For example, it has been found that zinc ions alone, when used at a zinc ion concentration of 1.16g/L, exhibit a toxic effect on osteoblasts. While when compounded with magnesium ions, 1.16g/L zinc ion toxicity was significantly inhibited, showing good bioactivity, as was the case with magnesium ions (see FIG. 5). This synergy results from the difference in the crosslinking characteristics and stability of the zinc and magnesium ions and the G units in the alginate. It is found that after two ions enter the alginate gel, the G unit eggshell-like structure formed by zinc ions and magnesium ions in the alginate gel is different, and the release rates of the two ions are delayed from each other, so that the free state concentration of the two ions is reduced, and the toxic effect of the two ions on cells is further reduced. Meanwhile, the action mechanisms of the two ions on the biological activity are different, so that a certain synergistic effect can be generated between the two ions after the two ions are mixed, and further the toxicity and release of the ions between the two ions are changed.
Therefore, a metal ion composite crosslinking system of the alginate bone tissue engineering scaffold is finally constructed.
Drawings
FIG. 1 scanning electron microscope of the internal structure of alginate gel under the action of different ionic crosslinking agents. (A) 4g/L calcium ion; (B) 4g/L calcium ion+4 g/L strontium ion; (C) 4g/L calcium ion+1.16 g/L zinc ion; (D) 4g/L calcium ion+2.44 g/L magnesium ion; (D) 4g/L calcium ion +4g/L strontium ion +1.16g/L zinc ion +2.44g/L magnesium ion.
FIG. 2 mechanical strength of alginate gels with different ionic cross-linking agents. (A) 4g/L calcium ion; (B) 4g/L calcium ion+4 g/L strontium ion; (C) 4g/L calcium ion+1.16 g/L zinc ion; (D) 4g/L calcium ion+2.44 g/L magnesium ion; (E) 4g/L calcium ion +4g/L strontium ion +1.16g/L zinc ion +2.44g/L magnesium ion.
FIG. 3 biological safety of alginate gels with different concentrations of zinc ion cross-linker. (A) 4g/L calcium ion+4 g/L strontium ion+0.7 g/L zinc ion; (B) 4g/L calcium ion+4 g/L strontium ion+1.16 g/L zinc ion; (C) 4g/L calcium ion+4 g/L strontium ion+2 g/L zinc ion.
FIG. 4 biological safety of alginate gels with varying concentrations of magnesium cross-linker. (A) 4g/L calcium ion+4 g/L strontium ion; (B) 4g/L calcium ion+4 g/L strontium ion+1 g/L magnesium ion; (C) 4g/L calcium ion+4 g/L strontium ion+2 g/L magnesium ion; (D) 4g/L calcium ion+4 g/L strontium ion+2.44 g/L magnesium ion.
FIG. 5 scanning electron microscope after adhesion of alginate gel osteoblasts under the action of different ionic crosslinking agents. (A) 4g/L calcium ion, 4g/L strontium ion, 0.35g/L zinc ion and 2g/L magnesium ion; (B) 4g/L calcium ion, 4g/L strontium ion, 1.16g/L zinc ion and 2g/L magnesium ion; (C) 4g/L calcium ion, 4g/L strontium ion, 0.7g/L zinc ion and 2.44g/L magnesium ion; (D) 4g/L calcium ion, 4g/L strontium ion, 2g/L zinc ion and 2g/L magnesium ion; (E) 4g/L calcium ion +4g/L strontium ion +1.16g/L zinc ion +2.44g/L magnesium ion.
FIG. 6 alginate gel osteoblast alkaline phosphatase activity under the influence of different ionic cross-linking agents. (A) 4g/L calcium ion, 4g/L strontium ion, 0.7g/L zinc ion and 2g/L magnesium ion; (B) 4g/L calcium ion, 4g/L strontium ion, 1.16g/L zinc ion and 2g/L magnesium ion; (C) 4g/L calcium ion, 4g/L strontium ion, 0.7g/L zinc ion and 2.44g/L magnesium ion; (D) 4g/L calcium ion, 4g/L strontium ion, 2g/L zinc ion and 2g/L magnesium ion; (E) 4g/L calcium ion +4g/L strontium ion +1.16g/L zinc ion +2.44g/L magnesium ion.
Detailed Description
The construction method of the metal ion composite crosslinking system of the alginate bone tissue engineering scaffold is characterized by comprising the following steps:
(1) Purification of alginate material: alginate with average molecular weight 4000 was added to triple distilled deionized water to prepare a solution with a concentration of 8% wt; placing a regenerated cellulose dialysis bag with the molecular weight cut-off of 3500 in 2% W/V sodium bicarbonate and 1mM EDTA with the pH of 8.0, boiling for 10 minutes, and thoroughly cleaning with distilled water; adding the alginate solution into a treated dialysis bag, sealing two ends of the dialysis bag, and placing the dialysis bag into a beaker; adding deionized water to submerge the dialysis bag, placing on a magnetic stirrer to stir for 72 hours, replacing the deionized water every 4 hours in the first 12 hours, and then replacing every 12 hours; after dialysis, collecting the solution in the dialysis bag, and freeze-drying for 24 hours to obtain alginate powder;
(2) Preparation of alginate precursor solution: weighing 4g of alginate powder and adding the alginate powder into a reagent bottle containing 100mL of 0.01M PBS solution, and placing the reagent bottle on a magnetic stirrer to stir for 12 hours so as to completely dissolve the alginate powder; titrating the alginate precursor solution to a pH of 7.4 using 0.1M dilute hydrochloric acid or sodium hydroxide solution, preparing an alginate solution having a final concentration of 4% wt, and filter-sterilizing the alginate solution using a 0.22 μm sterile filter;
(3) Configuration of the polyion composite crosslinking agent: weighing anhydrous CaCl 2 0.4g，SrCl 2 0.4g，ZnCl 2 0.116g and Mg Cl 2 0.244g, dissolving in 100mL double distilled water to prepare a mixed solution, enabling the concentration of the mixed solution to be 2 times of the final concentration of the cross-linking agent to be prepared, sterilizing the mixed solution by using a 0.22 mu m sterile filter, storing in a sterile reagent bottle, and uniformly mixing by spiral oscillation before use;
(4) Alginate gel synthesis: heating the alginate solution obtained in the step 2 to 60 ℃ before each use, magnetically stirring for 1h, and placing in a 37 ℃ water bath constant temperature box; and (3) respectively taking the alginate precursor solution with equal volume and the composite cross-linking agent obtained in the step (3), fully and uniformly mixing the alginate precursor solution and the composite cross-linking agent by using a sterile medical three-way pipe, injecting the mixture into any designated position within 10 seconds, and forming solid gel after 1 minute.
The effect of the composite crosslinking system of the present invention is described below with reference to the accompanying drawings:
FIG. 1 scanning electron microscope of the internal structure of alginate gel under the action of different ionic crosslinking agents. (A) 4g/L calcium ion; (B) 4g/L calcium ion+4 g/L strontium ion; (C) 4g/L calcium ion+1.16 g/L zinc ion; (D) 4g/L calcium ion+2.44 g/L magnesium ion; (D) 4g/L calcium ion +4g/L strontium ion +1.16g/L zinc ion +2.44g/L magnesium ion.
When the internal structure of the gel is observed by using an electron microscope, the gel structure is more uniform when 4g/L calcium ions, 4g/L strontium ions, 1.16g/L zinc ions and 2.44g/L magnesium ions are used as the composite cross-linking agent, the strength and stability of the gel are improved, and the gap difference of the gel structure formed by the other four ion cross-linking agents is large, so that the stability is poor.
FIG. 2 mechanical strength of alginate gels with different ionic cross-linking agents. (A) 4g/L calcium ion; (B) 4g/L calcium ion+4 g/L strontium ion; (C) 4g/L calcium ion+1.16 g/L zinc ion; (D) 4g/L calcium ion+2.44 g/L magnesium ion; (E) 4g/L calcium ion +4g/L strontium ion +1.16g/L zinc ion +2.44g/L magnesium ion.
The result shows that when 4g/L calcium ions, 4g/L strontium ions, 1.16g/L zinc ions and 2.44g/L magnesium ions are used as the composite cross-linking agent, the mechanical strength of the gel is obviously improved, which proves that the gel stability is obviously improved under the action of the cross-linking agent system.
FIG. 3 biological safety of alginate gels with different concentrations of zinc ion cross-linker. (A) 4g/L calcium ion+4 g/L strontium ion+0.7 g/L zinc ion; (B) 4g/L calcium ion+4 g/L strontium ion+1.16 g/L zinc ion; (C) 4g/L calcium ion+4 g/L strontium ion+2 g/L zinc ion.
The zinc ion concentration was selected based on the adhesion and proliferation of osteoblasts to the gel surface. The results show that when the concentration of zinc ions in the cross-linking agent is 0.7g/L zinc ions, the osteoblasts have optimal adhesion and proliferation effects on the surface of the gel. While a zinc ion concentration of 1.16g/L has a certain toxic effect on cells. When the zinc ion concentration reached 2g/L, cytotoxicity was significantly increased, with only a small number of cells surviving.
FIG. 4 biological safety of alginate gels with varying concentrations of magnesium cross-linker. (A) 4g/L calcium ion+4 g/L strontium ion; (B) 4g/L calcium ion+4 g/L strontium ion+1 g/L magnesium ion; (C) 4g/L calcium ion+4 g/L strontium ion+2 g/L magnesium ion; (D) 4g/L calcium ion+4 g/L strontium ion+2.44 g/L magnesium ion.
According to the adhesion and proliferation condition of osteoblasts on the gel surface, the proper magnesium ion concentration is subjected to preliminary screening. The results show that when the concentration of magnesium ions in the crosslinking agent is 2g/L, the osteoblasts have the best adhesion and proliferation effects on the surface of the gel. And when the concentration of magnesium ions in the crosslinking agent is 2.44g/L, the excessive concentration of magnesium ions has a certain toxic effect on cells.
FIG. 5 scanning electron microscope after adhesion of alginate gel osteoblasts under the action of different ionic crosslinking agents. (A) 4g/L calcium ion, 4g/L strontium ion, 0.35g/L zinc ion and 2g/L magnesium ion; (B) 4g/L calcium ion, 4g/L strontium ion, 1.16g/L zinc ion and 2g/L magnesium ion; (C) 4g/L calcium ion, 4g/L strontium ion, 0.7g/L zinc ion and 2.44g/L magnesium ion; (D) 4g/L calcium ion, 4g/L strontium ion, 2g/L zinc ion and 2g/L magnesium ion; (E) 4g/L calcium ion +4g/L strontium ion +1.16g/L zinc ion +2.44g/L magnesium ion.
And (3) carrying out scanning electron microscope observation on gel osteoblast adhesion after the combination and crosslinking of different types of ions. The results show that when strontium ions, zinc ions and magnesium ions with proper concentrations are respectively added, the cell adhesion is obviously improved. However, when four ions are combined to form a four metal ion composite cross-linking system, the number of adhered cells of the gel is significantly increased, indicating that the bioactivity of the alginate gel can be significantly improved when the composite cross-linking agent of the formulation is used.
In particular, zinc ions and magnesium ions have positive synergistic effect and reduce toxicity. As described above, after two ions enter the alginate gel, the G unit eggshell-like structure formed by the zinc ions and the magnesium ions in the alginate gel is different, and the two ions delay the release rate of each other, so that the free state concentration of the two ions is reduced, and the toxic effect of the two ions on cells is further reduced, which is also a phenomenon found in the reverse thinking experiment of the invention, and the addition of four particles at the same time according to a proper proportion does not only result in the increase of toxicity, but also results in better use effect due to forward synergy. For example, in the case of zinc ion alone, when the zinc ion concentration reaches 1.16g/L, a certain toxic effect on osteoblasts is exhibited. When the zinc ion is compounded with magnesium ions, the toxicity of 1.16g/L zinc ions is obviously inhibited, and the zinc ion shows good biological activity.
FIG. 6 alginate gel osteoblast alkaline phosphatase activity under the influence of different ionic cross-linking agents. (A) 4g/L calcium ion, 4g/L strontium ion, 0.7g/L zinc ion and 2g/L magnesium ion; (B) 4g/L calcium ion, 4g/L strontium ion, 1.16g/L zinc ion and 2g/L magnesium ion; (C) 4g/L calcium ion, 4g/L strontium ion, 0.7g/L zinc ion and 2.44g/L magnesium ion; (D) 4g/L calcium ion, 4g/L strontium ion, 2g/L zinc ion and 2g/L magnesium ion; (E) 4g/L calcium ion +4g/L strontium ion +1.16g/L zinc ion +2.44g/L magnesium ion.
Alkaline phosphatase activity is an important indicator of osteoblast differentiation. The results show that the compound cross-linking agent with four ions at proper concentration can effectively improve the expression of alkaline phosphatase activity, thereby proving that the alginate gel under the formula can effectively promote the osteogenesis activity.
Claims (2)
1. The construction method of the metal ion composite crosslinking system of the alginate bone tissue engineering scaffold is characterized by comprising the following steps:
(1) Purification of alginate material: alginate with average molecular weight 4000 was added to triple distilled deionized water to prepare a solution with a concentration of 8% wt; placing a regenerated cellulose dialysis bag with the molecular weight cut-off of 3500 in 2% W/V sodium bicarbonate and 1mM EDTA with the pH of 8.0, boiling for 10 minutes, and thoroughly cleaning with distilled water; adding the alginate solution into a treated dialysis bag, sealing two ends of the dialysis bag, and placing the dialysis bag into a beaker; adding deionized water to submerge the dialysis bag, placing on a magnetic stirrer to stir for 72 hours, replacing the deionized water every 4 hours in the first 12 hours, and then replacing every 12 hours; after dialysis, collecting the solution in the dialysis bag, and freeze-drying for 24 hours to obtain alginate powder;
(2) Preparation of alginate precursor solution: weighing 4g of alginate powder and adding the alginate powder into a reagent bottle containing 100mL of 0.01M PBS solution, and placing the reagent bottle on a magnetic stirrer to stir for 12 hours so as to completely dissolve the alginate powder; titrating the alginate precursor solution to a pH of 7.4 using 0.1M dilute hydrochloric acid or sodium hydroxide solution, preparing an alginate solution having a final concentration of 4% wt, and filter-sterilizing the alginate solution using a 0.22 μm sterile filter;
(3) Configuration of the polyion composite crosslinking agent: weighing anhydrous CaCl 2 0.4g，SrCl 2 0.4g，ZnCl 2 0.116g and Mg Cl 2 0.244g, dissolving in 100mL double distilled water to prepare a mixed solution, enabling the concentration of the mixed solution to be 2 times of the final concentration of the cross-linking agent to be prepared, sterilizing the mixed solution by using a 0.22 mu m sterile filter, storing in a sterile reagent bottle, and uniformly mixing by spiral oscillation before use;
(4) Alginate gel synthesis: heating the alginate solution obtained in the step 2 to 60 ℃ before each use, magnetically stirring for 1h, and placing in a 37 ℃ water bath constant temperature box; and (3) respectively taking the alginate precursor solution with equal volume and the composite cross-linking agent obtained in the step (3), fully and uniformly mixing the alginate precursor solution and the composite cross-linking agent by using a sterile medical three-way pipe, injecting the mixture into any designated position within 10 seconds, and forming solid gel after 1 minute.
2. The method for constructing an alginate bone tissue engineering scaffold metal ion composite crosslinking system according to claim 1, wherein the alginate is sodium alginate.
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| US6497902B1 (en) * | 1999-12-01 | 2002-12-24 | The Regents Of The University Of Michigan | Ionically crosslinked hydrogels with adjustable gelation time |
| JP2012153633A (en) * | 2011-01-25 | 2012-08-16 | Dentoro Chem:Kk | Dental alginate impression material |
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| CN109096522A (en) * | 2018-06-25 | 2018-12-28 | 苏州大学 | It is a kind of with the medical plural gel of more biological functions, preparation method and applications |
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| US6497902B1 (en) * | 1999-12-01 | 2002-12-24 | The Regents Of The University Of Michigan | Ionically crosslinked hydrogels with adjustable gelation time |
| JP2012153633A (en) * | 2011-01-25 | 2012-08-16 | Dentoro Chem:Kk | Dental alginate impression material |
| CN106994133A (en) * | 2016-01-25 | 2017-08-01 | 吉林省莱沃医疗科技有限公司 | Calcium zinc ion is combined antibacterial alginate slow-release material and preparation method thereof |
| CN109096522A (en) * | 2018-06-25 | 2018-12-28 | 苏州大学 | It is a kind of with the medical plural gel of more biological functions, preparation method and applications |
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