CN113913961A - Mineralized collagen nanofiber doped with active elements and preparation method thereof - Google Patents
Mineralized collagen nanofiber doped with active elements and preparation method thereof Download PDFInfo
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Abstract
The application provides mineralized collagen nanofibers doped with active elements and a preparation method thereof, wherein the mineralized collagen nanofibers are composed of collagen fibers and needle-shaped hydroxyapatite crystals; part of the calcium element in the crystal lattice of the hydroxyapatite crystal is replaced by cations containing the active element, and/or part of the phosphate groups in the crystal lattice of the hydroxyapatite crystal is replaced by anions containing the active element. The preparation process is simple and feasible, and the strength and the rigidity of the original high polymer material can be obviously improved by the product obtained by compounding the obtained mineralized collagen nano fibers and the high polymer material (collagen, PCL and the like). In addition, the doping scheme of multiple active elements is adopted to meet the cell microenvironment for inhibiting tumor, resisting infection and regulating immunity provided by material degradation in the bone repair process. The method provides a good idea for the design and development of the polymer bone repair material, and brings greater economic benefits for human health and social development.
Description
Technical Field
The invention relates to but is not limited to the field of biomedical materials, in particular to but not limited to mineralized collagen nanofibers doped with active elements and a preparation method thereof.
Background
The natural bone is mainly composed of inorganic components and organic components, wherein the inorganic components are mainly low-crystalline Hydroxyapatite (HA), and the organic components are mainly composed of protein and polysaccharide substances. Compared with natural bone materials, the bionic material prepared in vitro has the advantages of sufficient sources and low immunogenicity. Calcium and phosphorus elements are selected as main components to simulate natural bone components, and even the bone materials which simulate the minimum structural units of the natural bone by an in vitro mineralization method are more, so that a better osteogenesis induction effect can be achieved.
However, in clinical needs, the repair of bone defects not only needs to consider the bone regeneration performance, but also has the key of having targeted additional functions in combination with the reason for causing bone defects and the postoperative repair needs. Besides the main components of calcium and phosphorus, natural bone also contains various trace elements, such as magnesium, silicon, zinc, etc. The introduction of the trace elements can not only improve the osteogenesis inducing capability of the material, but also realize various additional biological functions. However, the method for synthesizing microelement-doped mineralized collagen in the prior art is complex and tedious, and the obtained mineralized collagen which is not in a fiber form greatly limits the development and application of the artificially synthesized material.
Disclosure of Invention
The following is a summary of the subject matter described in detail herein. This summary is not intended to limit the scope of the present application.
The invention relates to a method for preparing mineralized collagen nano-fibers doped with multiple active elements by in-situ co-assembly, which is characterized in that collagen is assembled into a template on a molecular level to regulate in-situ deposition and co-assembly of nano calcium-phosphorus salt.
The application provides an active element doped mineralized collagen nanofiber, which consists of collagen fibers and needle-shaped hydroxyapatite crystals;
part of calcium in the crystal lattice of the hydroxyapatite is replaced by cations containing the active element,
and/or, part of the phosphate groups in the crystal lattice of the hydroxyapatite are replaced by anions containing the active element; the anion of the active element can be an oxyacid radical of the active element anion;
the active element comprises any one or more of silicon, selenium, magnesium, zinc, strontium, silver, iron (ferric iron) and copper (cupric copper);
the ratio of the stoichiometric number of the calcium element to the sum of the active element-containing cation and the phosphate radical and the active element-containing anion is (1.45:1) to (1.8: 1);
containing the molar ratio of the active element cation to the calcium ion (5 to 8) to (92 to 95); contains the molar ratio of the active element anion to the phosphate radical (5 to 7) to (93 to 95).
In one embodiment provided herein, the active element cation and the active element anion can be present in an amount of 0.
In one embodiment provided herein, the mineralized collagen nanofibers doped with active elements have an average length of 250nm to 300 nm;
in one embodiment provided herein, the mineralized collagen nanofibers doped with active elements have an average diameter of 5nm to 7 nm.
In another aspect, the present application provides a method for preparing the above mineralized collagen nanofibers doped with active elements, comprising: preparing the mineralized collagen nano fiber doped with the active element by using an in-situ co-assembly method, and adding cations containing the active element while adding calcium salt ions and/or adding anions containing the active element while adding phosphate ions.
In one embodiment provided herein, a method for preparing mineralized collagen nanofibers doped with active elements comprises the following steps:
(1) mixing the type I collagen sponge with a phosphoric acid solution to completely dissolve the type I collagen sponge to obtain a collagen template solution for later use;
when the active element is doped in the form of anion, firstly, the salt containing the anion is mixed with the phosphoric acid solution until the salt is completely dissolved, and then, the salt is mixed with the type I collagen sponge;
(2) preparing a calcium salt solution, wherein the calcium salt solution and the collagen template solution have the same volume;
when the active element is doped in the form of cation, firstly, the salt containing the cation is mixed with the calcium salt solution until the salt is completely dissolved, and then the salt is mixed with the collagen template solution;
(3) preparing a buffer solution;
(4) titrating the collagen template solution and the calcium salt solution in a buffer solution to obtain a mixed solution after titration is finished; during the titration, the pH value of the mixed solution is maintained between 8 and 10;
(5) carrying out high-speed centrifugation on the mixed solution obtained in the step (4), replacing the supernatant obtained by the centrifugation with ultrapure water with the same volume until the pH value of the measured supernatant is about 7-8, and stopping the high-speed centrifugation;
(6) removing water from the precipitate obtained in the step (5) to obtain mineralized collagen nanofibers doped with active elements;
alternatively, the preparation method consists of the above.
In one embodiment provided herein, the molar ratio of active element-containing cations to calcium ions is (5 to 10): (95 to 90);
in one embodiment provided herein, the molar ratio of active element-containing anions to calcium ions is (5 to 10): (95 to 90);
in one embodiment provided herein, the ratio of the stoichiometric numbers of (calcium ions + active element cations)/(phosphate ions + active element anions) is from (1.65:1) to (1.8: 1);
in one embodiment provided herein, the concentration of the phosphoric acid solution of step (1) is 0.5 to 2mol/L, and the volume ratio of the mass of the type I collagen sponge to the phosphoric acid solution is 0.1 to 10 g/L;
in one embodiment provided herein, the mixing time for the mixing of step (1) is 4 to 12 hours; optionally, the mixing temperature of the mixing is 35 ℃ to 38 ℃.
In one embodiment provided herein, the calcium salt solution in step (2) further comprises hydroxide, and the molar ratio of the sum of the calcium ions and the active element cations to the hydroxide is 2: 1.
In one embodiment provided herein, the buffer solution of step (3) is selected from any one or more of Tris-HCl and phosphate buffer solutions.
In one embodiment provided herein, the flow rate of the titration of step (4) is 400ml/h to 500 ml/h;
in one embodiment provided herein, after the titration in step (4) is completed, stirring for 12 to 36 hours; optionally, the reaction temperature is controlled at 30 ℃ to 37 ℃ after the titration is completed.
In one embodiment provided herein, the centrifugation of step (5) is performed at a speed of 3000 rpm to 17000 rpm.
In one embodiment provided herein, the removing of water in step (6) is low temperature lyophilization, comprising: drying the precipitate at 4-8 ℃, and then removing residual water by adopting freeze drying; optionally, the freeze dryer has a cold well temperature of between 2 ℃ and 4 ℃, a vacuum degree of between 10Pa and 30Pa, and a freeze drying duration of between 24h and 72 h.
In one embodiment provided herein, the resulting mixture is thoroughly mixed using a magnetic stirrer for agitation.
The mineralized collagen nanofiber doped with the active elements has good bone compatibility, bone conductivity and certain osteogenesis induction capacity. The material can be combined with the original bone tissue after being implanted, and meanwhile, the material can collect the osteogenesis related cells and promote the osteogenesis related cells to be differentiated in the osteogenesis direction, so that the bone regeneration is realized.
Meanwhile, the mineralized collagen nanofibers doped with the active elements provided by the application cannot shield X rays.
The application provides an active element doped mineralized collagen nanofiber structure, which can provide a multi-element strengthening effect in the field of bone repair.
In addition, the mineralized collagen nanofibers doped with active elements can be used as reinforcements of high polymer materials, and the long fiber structure can obviously improve the toughness and strength of materials such as fiber membranes. By controlling the degree of crystallinity, the effect between the degradation rate and the osteogenesis rate is matched.
Additional features and advantages of the application will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by the practice of the application. Other advantages of the present application may be realized and attained by the invention in its aspects as described in the specification.
Drawings
The accompanying drawings are included to provide an understanding of the present disclosure and are incorporated in and constitute a part of this specification, illustrate embodiments of the disclosure and together with the examples serve to explain the principles of the disclosure and not to limit the disclosure.
FIG. 1 shows mineralized collagen nanofiber powder doped with active elements prepared in the examples of the present application.
FIG. 2 shows the morphology of the active element doped mineralized collagen nanofibers by TEM electron microscopy. It can be seen from the figure that the magnification is 20 ten thousand, the scale is 20nm, and the final products all present independent fiber structures with lengths between 250nm and 300 nm. After different active elements are doped, the size of the mineralized collagen nano fibers is not changed.
Fig. 3a and 3b show SEM images of silicon-magnesium doped mineralized collagen nanofibers prepared in example 6. FIG. 3b is an enlarged view of selected regions of FIG. 3a, showing that the product obtained by the freeze-drying process is fibrous, and the structural morphology of the mineralized collagen nanofibers is not affected by the element incorporation.
Fig. 4 is a schematic diagram of EDS element distribution of the silicon-magnesium doped mineralized collagen nanofibers prepared in example 6. From the figure, it can be seen that Mg, Si element was successfully incorporated into the mineralized collagen nanofiber structure.
Fig. 5a, 5b, 5c, 5d, 5e, 5f and 5g are silicon-magnesium surface scanning element distribution diagrams of the mineralized collagen nanofibers doped with silicon-magnesium prepared in example 6. As can be seen from the figure, the silicon and magnesium elements are successfully introduced, and all show the doping morphology with overlapping regularity.
Fig. 6a and 6b show SEM images of strontium-doped mineralized collagen nanofibers prepared in example 1. FIG. 6b is an enlarged view of selected regions of FIG. 6a, showing that the product obtained by lyophilization is fibrous and the morphology of the mineralized collagen nanofiber structure is not affected by the element incorporation.
Fig. 7a, 7b, 7c, 7d, 7e and 7f are strontium element surface scanning element distribution diagrams of the mineralized collagen nanofibers doped with strontium element prepared in example 1. As can be seen from the figure, Sr element is successfully introduced, and the doping morphology with overlapping regularity is presented.
Fig. 8 is a stress-strain curve of the fiber membrane obtained in application example 2 and application example 3. It can be seen from the figure that the strength of the fiber membrane obtained by electrospinning the mineralized collagen nanofibers doped with silicon and magnesium is obviously improved compared with the mineralized collagen fibers prepared by the same method and not doped with silicon and magnesium.
Fig. 9 is a schematic illustration of a rat cranioplasty animal experiment with fibrous membranes containing strontium-doped mineralized collagen nanofibers provided in example 1 of the present application (the size of each set of membranes is the same). In the figure, contrast is a control group, collagen membrane is a common collagen membrane (a collagen fiber membrane prepared by electrostatic spinning), BLB is a fiber membrane prepared by application example 3, Sr substistuted BLB is a strontium-doped mineralized collagen nanofiber and collagen composite group prepared by application example 1, 2W is two weeks, and 3W is three weeks; it can be seen from the figure that the fibrous structure prepared in example 1 can promote bone repair by mixing with other high molecular structure materials. The addition of Sr element can promote the bone repairing process.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, embodiments of the present application are described in detail below. It should be noted that the embodiments and features of the embodiments in the present application may be arbitrarily combined with each other without conflict.
In the examples provided herein, the raw materials were sourced as follows: sodium silicate (Na)2SiO3Analytically pure), anhydrous calcium chloride (CaCl)2Analytically pure), sodium hydroxide (NaOH, analytically pure) and ammonia (NH)4OH analytical pure) was purchased from pharmaceutical chemicals, ltd; strontium chloride hexahydrate (SrCl)26H2O, 99.5%, available from Shanghai Aladdin Biotechnology Ltd; type I collagen sponge (atelocollagen from oxtail skin, MW 300,000 oxtail, north river collectison); phosphoric acid (H)3PO4Analytically pure, Shanghai Tantake Technique, Inc.); Tris-HCl (C)4H11NO3HCl, analytical pure, beijing baidi biotechnology limited).
Example 1
(1) Dissolving the type I collagen sponge in phosphoric acid ultrapure water solution, wherein the concentration of the phosphoric acid solution is 0.5mol/L, and the volume ratio of the mass of the type I collagen sponge to the phosphoric acid solution is 0.1g: 1L. And fully mixing the obtained mixture by using a magnetic stirrer for 6 hours to fully dissolve the added I-type collagen template, and marking as an anionic solution for later use. The temperature of the system is maintained between 35 and 38 ℃ during the stirring process.
(2) Preparing calcium chloride ultrapure water solution, adding CaCl according to the preset molar relative percentage of doped cation salt (Sr) of 9%2The solution is stirred and dissolved uniformly (i.e. the molar ratio of strontium ions to calcium ions is 9: 91).
Preparing an ultrapure aqueous NaOH solution, and ensuring that the molar ratio of (calcium ions + strontium ions)/OH is 2. Adding NaOH solution into calcium-containing strontium cation solution, and recording as cation solution for standby.
The volume ratio of the prepared cation solution to the solution prepared in the step (1) is 1: 1. The total amount ensures that the ratio of (calcium + strontium ion)/(phosphate ion) stoichiometric number is 1.65: 1.
(3) Tris-HCl buffer solution with the solubility of 1mol/L is prepared, namely 0.2mol of Tris-HCl is weighed and dissolved in 200ml of ultrapure water to be used as reaction kettle liquid. And adjusting the pH value of the reaction kettle liquid to be between 8 and 10 by using 0.5mol/L ammonia water and HCl solution.
(4) Simultaneously titrating the anion solution and the cation solution in the reaction kettle liquid, controlling the flow rate to be maintained at 500ml/h, ensuring that the pH value of the reaction kettle liquid is maintained between 8 and 10 in the titration process, controlling the reaction temperature to be 37 ℃ after the titration is finished, and fully stirring for 16h in a dark place. And finishing titration of the anion solution and the cation solution with equal volumes.
(5) And (5) transferring the system in the step (4) into a centrifugal tube for high-speed centrifugation. And after each centrifugation, collecting the supernatant, measuring the pH value of the supernatant, adding ultrapure water with the same volume as the supernatant into the centrifuge tube, stirring the precipitate by using a glass rod to make the precipitate fully contact with the ultrapure water so as to clean the precipitate, and centrifuging again. This was repeated two to three times (3000 rpm) until the pH of the eluate was measured at around 7 to 8; finally 17000 r/min to obtain the cleaned precipitate.
(6) Drying the precipitate obtained after cleaning at 4 deg.C, and removing residual water by freeze dryer with freeze well temperature of 4 deg.C, vacuum degree of 30Pa, and freeze drying duration of 50 hr or more. And drying to obtain the strontium-doped mineralized collagen nanofiber.
The mineralized collagen nanofiber doped with strontium prepared in this example is shown in fig. 6, and it can be seen from fig. 7 that calcium ions in the crystal lattice of hydroxyapatite are replaced by Sr element.
As shown in table 1, the ratio of the sum of the stoichiometric numbers of calcium ions and strontium ions to the stoichiometric number of phosphate anions in the strontium-doped mineralized collagen nanofibers prepared in this example was 1.80: 1.
As shown in table 1, the molar ratio of strontium ions to the calcium ions in the strontium-doped mineralized collagen nanofibers prepared in this example was 6: 94.
The strontium-doped mineralized collagen nanofibers prepared by the present example had an average length of 250nm to 300nm and an average diameter of 5nm to 7 nm.
Example 2
(1) Dissolving the type I collagen sponge in phosphoric acid ultrapure water solution, wherein the concentration of the phosphoric acid solution is 2mol/L, and the volume ratio of the mass of the type I collagen sponge to the phosphoric acid solution is 10g: 1L. And fully mixing the obtained mixture by using a magnetic stirrer for 12 hours, so that the added I-type collagen template is fully dissolved and marked as an anionic solution for later use. The temperature of the system was maintained between 35 ℃ and 38 ℃ during stirring.
(2) Preparing calcium chloride ultrapure water solution, adding CaCl according to the preset doped cation salt (Sr) molar relative percentage of 7%2The solution is stirred and dissolved uniformly (i.e. the molar ratio of strontium ions to calcium ions is 7: 93).
Preparing an ultrapure aqueous NaOH solution, and ensuring that the molar ratio of (calcium ions + strontium ions)/OH is 2. Adding NaOH solution into calcium-containing strontium cation solution, and recording as cation solution for standby.
The volume ratio of the prepared cation solution to the solution prepared in the step (1) is 1: 1. The total amount ensures that the ratio of (calcium + doped cation)/(phosphate) stoichiometric number is 1.67: 1.
(3) Tris-HCl buffer solution with the solubility of 5mol/L is prepared, namely 1.0mol of Tris-HCl is weighed and dissolved in 200ml of ultrapure water to be used as reaction kettle liquid. And adjusting the pH value of the reaction kettle liquid to be between 8 and 10 by using 0.5mol/L ammonia water and HCl solution.
(4) Simultaneously titrating the anion solution and the cation solution in the reaction kettle liquid, controlling the flow rate to be maintained at 500ml/h, ensuring that the pH value of the reaction kettle liquid is maintained between 8 and 10 in the titration process, controlling the reaction temperature to be 37 ℃ after the titration is finished, and fully stirring for 36h in a dark reaction. And finishing titration of the anion solution and the cation solution with equal volumes.
(5) And (5) transferring the system in the step (4) into a centrifugal tube for high-speed centrifugation. And after each centrifugation, collecting the supernatant, measuring the pH value of the supernatant, adding ultrapure water with the same volume as the supernatant into the centrifuge tube, stirring the precipitate by using a glass rod to make the precipitate fully contact with the ultrapure water so as to clean the precipitate, and centrifuging again. This was repeated two to three times (3000 rpm) until the pH of the eluate was measured at around 7 to 8; finally 17000 r/min to obtain the cleaned precipitate.
(6) Drying the precipitate obtained after cleaning at 4 deg.C, and removing residual water by freeze dryer with freeze well temperature of 4 deg.C, vacuum degree of 30Pa, and freeze drying duration of 50 hr or more.
In the strontium-doped mineralized collagen nanofibers prepared by the embodiment, the ratio of the sum of the stoichiometric numbers of calcium ions and strontium ions to the stoichiometric number of phosphate radicals is 1.67: 1;
the molar ratio of strontium ions to the calcium ions in the strontium-doped mineralized collagen nanofibers prepared by the embodiment is 6: 94;
the strontium-doped mineralized collagen nanofibers prepared by the present example had an average length of 250nm to 300nm and an average diameter of 5nm to 7 nm.
Example 3
(1) Dissolving the type I collagen sponge in phosphoric acid ultrapure water solution, wherein the concentration of the phosphoric acid solution is 1mol/L, and the volume ratio of the mass of the type I collagen sponge to the phosphoric acid solution is 2g: 1L. And fully mixing the obtained mixture by using a magnetic stirrer for 9 hours to fully dissolve the added I-type collagen template, and marking as an anionic solution for later use. The temperature of the system was maintained between 35 ℃ and 38 ℃ during stirring.
(2) Preparing calcium chloride ultrapure water solution, adding CaCl according to the preset doped cation salt (Sr) molar relative percentage of 7%2The solution is stirred and dissolved uniformly (i.e. the molar ratio of strontium ions to calcium ions is 7: 93).
Preparing an ultrapure aqueous NaOH solution, and ensuring that the molar ratio of (calcium ions + strontium ions)/OH is 2. Adding NaOH solution into calcium-containing strontium cation solution, and recording as cation solution for standby.
The volume ratio of the prepared cation solution to the solution prepared in the step (1) is 1: 1. The total amount ensures that the ratio of (calcium + strontium ion)/(phosphate ion) stoichiometric number is 1.67: 1.
(3) Tris-HCl buffer solution with the solubility of 3mol/L is prepared, namely 0.6mol of Tris-HCl is weighed and dissolved in 200ml of ultrapure water to be used as reaction kettle liquid. And adjusting the pH value of the reaction kettle liquid to be between 8 and 10 by using 0.5mol/L ammonia water and HCl solution.
(4) Simultaneously titrating the anion solution and the cation solution in the reaction kettle liquid, controlling the flow rate to be maintained at 450ml/h, ensuring that the pH value of the reaction kettle liquid is maintained between 8 and 10 in the titration process, controlling the reaction temperature to be 37 ℃ after the titration is finished, and fully stirring for 24h in a dark place. And finishing titration of the anion solution and the cation solution with equal volumes.
(5) And (5) transferring the system in the step (4) into a centrifugal tube for high-speed centrifugation. And after each centrifugation, collecting the supernatant, measuring the pH value of the supernatant, adding ultrapure water with the same volume as the supernatant into the centrifuge tube, stirring the precipitate by using a glass rod to make the precipitate fully contact with the ultrapure water so as to clean the precipitate, and centrifuging again. This was repeated two to three times (3000 rpm) until the pH of the eluate was measured at around 7 to 8; finally 17000 r/min to obtain the cleaned precipitate.
(6) Drying the precipitate obtained after cleaning at 4 deg.C, and removing residual water by freeze dryer with freeze well temperature of 4 deg.C, vacuum degree of 30Pa, and freeze drying duration of 50 hr or more. And drying to obtain the strontium-doped nano mineralized collagen.
In the strontium-doped mineralized collagen nanofibers prepared by the embodiment, the ratio of the stoichiometric number of calcium ions and magnesium ions to the stoichiometric number of phosphate radicals is 1.65: 1;
in the strontium-doped mineralized collagen nanofibers prepared by the embodiment, the molar ratio of strontium ions to calcium ions is 6: 94;
the strontium-doped mineralized collagen nanofibers prepared by the present example had an average length of 250nm to 300nm and an average diameter of 5nm to 7 nm.
Example 4
This example differs from example 1 only in that:
the active element Mg replaces Sr, and the using amount and other preparation processes are completely the same as those of the example 1.
In the mineralized collagen nanofiber doped with magnesium prepared in the embodiment, the ratio of the stoichiometric number of calcium ions and magnesium ions to the stoichiometric number of phosphate radicals is 1.55: 1;
the molar ratio of magnesium ions to the calcium ions in the mineralized collagen nanofibers doped with magnesium prepared in the embodiment is 7.8: 92.2;
the mineralized collagen nanofibers doped with magnesium prepared in this example had an average length of 250nm to 300nm and an average diameter of 5nm to 7 nm.
Example 5
This example differs from example 1 only in that:
the active element is Si, and Sr is not involved;
(1) doping with anion salt (sodium silicate Na) according to preset value2SiO3) Adding the phosphoric acid into the phosphoric acid ultrapure water solution according to the relative mol percentage of 6 percent, and uniformly stirring. And dissolving the I-type collagen sponge in the phosphoric acid ultrapure water solution, wherein the concentration of the phosphoric acid solution is 0.5mol/L, and the volume ratio of the mass of the I-type collagen sponge to the phosphoric acid solution is 0.1g: 1L. And fully mixing the obtained mixture by using a magnetic stirrer for 6 hours to fully dissolve the added I-type collagen template, and marking as an anionic solution for later use. The temperature of the system was maintained between 35 ℃ and 38 ℃ during stirring.
In the present embodiment, step (2) does not involve active element Sr, and NaOH ultrapure water solution is prepared to ensure that (calcium ion)/OH is 2. Adding NaOH solution into calcium-containing cation solution, and recording as cation solution for standby. The other steps are the same as in example 1.
The ratio of the sum of the stoichiometric numbers of calcium ions to the stoichiometric number of phosphate anions in the silicon-doped mineralized collagen nanofibers prepared by the embodiment is 1.67: 1;
the molar ratio of silicate to phosphate in the silicon-doped mineralized collagen nanofibers prepared by the embodiment is 5: 95;
the mineralized collagen nanofibers doped with silicon prepared in this example had a mean diameter of 5nm to 7nm, and a diameter of 250nm to 300 nm.
Example 6
This example differs from example 5 only in that it contains Mg cation doping and Si silicate anion doping.
The procedure in this exampleStep (2) introduces magnesium ions: (2) preparing calcium chloride ultrapure water solution, adding CaCl according to the preset doped cation salt (Mg) molar relative percentage of 9%2The solution is stirred and dissolved uniformly.
Preparing an ultrapure aqueous NaOH solution, and ensuring that the molar ratio of (calcium ions + magnesium ions)/OH is 2. Adding NaOH solution into calcium-containing magnesium cation solution, and recording as cation solution for standby.
As shown in table 2, the ratio of the stoichiometric numbers of (calcium ion + magnesium ion)/(phosphate ion + silicate ion) in the mineralized collagen nanofibers produced in this example was 1.45: 1.
As shown in table 2, the molar ratio of silicate to phosphate in the mineralized collagen nanofibers doped with magnesium and silicon prepared in this example is 5: 95;
as shown in table 2, the molar ratio of magnesium ions to calcium ions in the mineralized collagen nanofibers doped with magnesium and silicon prepared in this example is 7.8: 92.2;
the mineralized collagen nanofibers doped with magnesium and silicon prepared by the embodiment have the average length of 250nm to 300nm and the average diameter of 5nm to 7 nm.
Comparative example 1
This comparative example differs from example 1 only in that:
this comparative example does not involve Sr element doping, and other raw materials and preparation methods are the same as those of example 1.
Table 1 elemental distribution of strontium-doped mineralized collagen nanofibers obtained in example 1
| Element(s) | Mass percentage of | Atomic number percentage |
| C | 12.64 | 24.35 |
| O | 45.13 | 55.06 |
| Sr | 1.62 | 0.79 |
| P | 15.06 | 7.36 |
| Ca | 25.55 | 12.44 |
| Total of | 100.00 | 100 |
Table 2 distribution of element content in mg-si doped mineralized collagen nanofibers prepared in example 6
| Element(s) | Mass percentage of | Atomic number percentage |
| C | 28.24 | 39.55 |
| O | 46.11 | 47.31 |
| Mg | 0.86 | 0.61 |
| Si | 0.5 | 0.27 |
| P | 9.41 | 5.08 |
| Ca | 14.88 | 7.18 |
| Total of | 100.00 | 100.00 |
Application example 1
The preparation of the fibrous membrane using the strontium-doped mineralized collagen nanofibers prepared in example 1 as the raw material in this application example comprises the following steps:
(1) the strontium-doped mineralized collagen nanofibers prepared in example 1 were taken, mixed with collagen and hexafluoroisopropanol, and continuously stirred vigorously for 28h to ensure thorough and uniform mixing. The mass ratio of the strontium doped mineralized collagen nanofibers, collagen and hexafluoroisopropanol solution prepared in example 1 was 1.0:0.6: 10.
(2) By electrospinning, a continuous fiber membrane having a certain thickness (the thickness may be 20 μm to 200 μm as needed) is formed. The electrospinning process can be referred to as "[ 1] Limonite, Huang Gong Ming. electrospinning of polymers [ J ]. macromolecules bulletin, 2006(05): 12-19" or as conventional in the art electrospinning process.
(3) And (3) drying the membrane prepared in the step (2) in a drying oven at 25 ℃ for 24 hours to ensure that residual trace hexafluoroisopropanol is removed. And then overlapping the multilayer films, and performing isostatic pressing at 3MPa for 5min by using a tablet press to obtain the modified fiber film with the multilayer structure.
(4) The method for preparing the EDC-NHS crosslinking solution by crosslinking a film with a certain thickness obtained under a certain pressure by using EDC-NHS comprises the following steps: EDC with the final concentration of 5mol/L and LNHS with the final concentration of 10mol/L are prepared and dissolved in a solvent, the solvent consists of 90% alcohol and 10% ultrapure water, the crosslinking is carried out at the low temperature of 4 ℃ for 2h, and the crosslinked film can not be layered.
(5) And cleaning the crosslinked modified fiber membrane by using alcohol to remove unreacted EDC, NHS and other impurity phases. And then the mixture is dried and stored in vacuum at 25 ℃, and finally the fibrous membrane of the strontium-doped mineralized collagen nanofiber is obtained.
In the fibrous membrane prepared by the application example, the inorganic substance in the mineralized collagen nano fibers doped with strontium accounts for 24.5 percent of the mass ratio of the whole fibrous membrane.
The porosity of the fiber membrane prepared by the application example is 80 percent;
the density of the fiber membrane prepared by the application example is 1.3g/cm3。
Application example 2
Application example 2 differs from application example 1 only in that: the mineralized collagen nanofibers doped with magnesium and silicon prepared in example 6 were used. Other preparation processes, raw materials and the use amount are the same as those of the application example 1.
In the fiber membrane prepared by the application example, the mass of inorganic matters in the mineralized collagen nano fibers doped with magnesium and silicon accounts for 24.5 percent of the whole fiber membrane;
the porosity of the fiber membrane prepared by the application example is 80 percent;
the density of the fiber membrane prepared by the application example is 1.3g/cm3。
The molar ratio of the sum of the calcium element and the magnesium element to the sum of the phosphorus element and the silicon element in the fiber film is 1.64: 1.
Application example 3
Application example 3 the mineralized collagen nanofibers doped with inactive elements prepared in comparative example 1 were used to prepare a fibrous membrane, and the preparation process, other raw materials and amounts were the same as in example 3.
The porosity of the fiber membrane prepared by the application example is 80 percent;
the density of the fiber membrane prepared by the application example is 1.3g/cm3。
As can be seen from fig. 8 and 9, the mineralized collagen nanofibers doped with active elements provided by the present application can improve osteogenesis inducing ability of the material, provide a multi-element strengthening effect in the bone repair field, and can significantly increase toughness and strength of the material. In addition, the doping scheme of multiple active elements can also meet the requirement of providing a cell microenvironment for inhibiting tumor, resisting infection and immunoregulation when the material is degraded in the bone repair process; and adjusting the crystallinity of the mineralized collagen nanofibers and maintaining the fiber structure of 250nm to 300nm to the maximum extent. The crystallinity state is adjusted to meet the requirement that the degradation rate of the bone repair scaffold material in vivo is matched with the growth speed of new bone.
Although the embodiments disclosed in the present application are described above, the descriptions are only for the convenience of understanding the present application, and are not intended to limit the present application. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the disclosure as defined by the appended claims.
Claims (10)
1. An active element doped mineralized collagen nanofiber, which consists of collagen fibers and needle-shaped hydroxyapatite crystals;
optionally, the calcium element in the crystal lattice of the hydroxyapatite is replaced by a cation containing the active element,
and/or the phosphate groups in the crystal lattice of the hydroxyapatite are replaced by anions containing the active element;
optionally, the active element comprises any one or more of silicon, selenium, magnesium, zinc, strontium, silver, iron and copper;
optionally, the ratio of elemental calcium to the stoichiometric number containing the active element cation to the stoichiometric number of the sum of the phosphate and the active element-containing anion is from (1.45:1) to (1.80: 1);
optionally, a molar ratio (5 to 8) of active element cations to calcium ions (92 to 95); contains the molar ratio of the active element anion to the phosphate radical (5 to 7) to (93 to 95).
2. The active element-doped mineralized collagen nanofibers according to claim 1, wherein the active element-doped mineralized collagen nanofibers have an average length of 250nm to 300 nm;
optionally, the active element-doped mineralized collagen nanofibers have an average diameter of 5nm to 7 nm.
3. The method for preparing mineralized collagen nanofibers doped with active elements according to claim 1 or 2, comprising: preparing the mineralized collagen nano fiber doped with the active element by using an in-situ co-assembly method, and adding cations containing the active element while adding calcium salt ions and/or adding anions containing the active element while adding phosphate ions.
4. The method for preparing mineralized collagen nanofibers doped with active elements according to claim 3, comprising the following steps:
(1) mixing the type I collagen sponge with a phosphoric acid solution to completely dissolve the type I collagen sponge to obtain a collagen template solution for later use;
when the active element is doped in the form of anion, firstly, the salt containing the anion is mixed with the phosphoric acid solution until the salt is completely dissolved, and then, the salt is mixed with the type I collagen sponge;
(2) preparing a calcium salt solution, wherein the calcium salt solution and the collagen template solution have the same volume;
when the active element is doped in the form of cation, firstly, the salt containing the cation is mixed with the calcium salt solution until the salt is completely dissolved, and then the salt is mixed with the collagen template solution;
(3) preparing a buffer solution;
(4) titrating the collagen template solution and the calcium salt solution in a buffer solution to obtain a mixed solution after titration is finished; during the titration, the pH value of the mixed solution is maintained between 8 and 10;
(5) carrying out high-speed centrifugation on the mixed solution obtained in the step (4), replacing the supernatant obtained by the centrifugation with ultrapure water with the same volume until the pH value of the measured supernatant is about 7-8, and stopping the high-speed centrifugation;
(6) and (5) removing the water in the precipitate obtained in the step (5) to obtain the mineralized collagen nanofiber doped with the active elements.
5. The method for preparing mineralized collagen nanofibers doped with active elements according to claim 4, wherein the concentration of the phosphoric acid solution in step (1) is 0.5 to 2mol/L, and the ratio of the mass of type I collagen sponge to the volume of the phosphoric acid solution is 0.1 to 10 g/L;
optionally, the mixing time of the mixing of step (1) is 4 to 12 hours; optionally, the mixing temperature of the mixing is 35 ℃ to 38 ℃.
6. The method for preparing mineralized collagen nanofibers doped with active elements according to claim 4, wherein the calcium salt solution in step (2) further comprises hydroxyl, and the molar ratio of the sum of calcium ions and the active element cations to the hydroxyl is 2: 1.
7. The method for preparing mineralized collagen nanofibers doped with active elements according to any one of claims 4 to 6, wherein the buffer solution of step (3) is selected from any one or more of Tris-HCl and phosphate buffer solution.
8. The method for preparing mineralized collagen nanofibers doped with active elements according to any one of claims 4 to 6, wherein the flow rate for the titration of step (4) is 400ml/h to 500 ml/h;
optionally, after the titration in the step (4) is completed, stirring for 12 to 36 hours; optionally, the reaction temperature is controlled at 30 ℃ to 37 ℃ after the titration is completed.
9. The method for preparing mineralized collagen nanofibers doped with active elements according to any one of claims 4 to 6, wherein the high speed centrifugation speed of step (5) is 3000 rpm to 17000 rpm.
10. The method for preparing mineralized collagen nanofibers doped with active elements according to any one of claims 4 to 6, wherein the removing of water in step (6) is low temperature lyophilization comprising: drying the precipitate at 4-8 ℃, and then removing residual water by adopting freeze drying; optionally, the freeze well temperature of the freeze dryer is between 2 ℃ and 4 ℃, the vacuum degree is 10Pa to 30Pa, and the freeze drying duration is 24h to 72 h.
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