CN115054733B - Preparation method and application of silk fibroin/hydroxyapatite composite micro-nano particle material - Google Patents
Preparation method and application of silk fibroin/hydroxyapatite composite micro-nano particle material Download PDFInfo
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Abstract
The invention provides a preparation method of a silk fibroin/hydroxyapatite composite micro-nano particle material, which comprises the steps of sequentially soaking silk fibroin nano particle powder in a solution containing calcium ions and phosphate ions to carry out auxiliary mineralization reaction, wherein nucleation sites are generated on the surfaces of the silk fibroin nano particles in the auxiliary mineralization reaction process, so that the growth of hydroxyapatite on the silk fibroin nano particles can be promoted, and meanwhile, the subsequent mineralization process is facilitated; then, the silk fibroin nanoparticle powder after auxiliary mineralization is soaked in simulated body fluid for mineralization reaction, so that hydroxyapatite crystals can be formed on the silk fibroin nanoparticle powder by self-assembly, and the silk fibroin/hydroxyapatite composite micro-nanoparticle material is finally prepared after freeze drying. The silk fibroin/hydroxyapatite composite micro-nano particle material prepared by the invention can be directly applied to human bodies, and has the characteristics of good biocompatibility, good stability and capability of inducing bone repair.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a preparation method and application of a silk fibroin/hydroxyapatite composite micro-nano particle material.
Background
Hydroxyapatite occupies a large part of human bone, wherein the human bone contains 60% of hydroxyapatite, 97% of enamel and 70% of dentin. The hydroxyapatite is used for biomedical materials due to the excellent biological performance, and because the ingredients and the structures of the hydroxyapatite and the natural rod-shaped nano hydroxyapatite in the enamel are almost the same and are easy to be compatible with the enamel/dentin, the hydroxyapatite can directly replace lost mineral substances, fill microscopic cracks on the surface of the enamel, promote natural healing of microscopic damage of the tooth, restore the smoothness of the surface of the tooth and is also used as an oral care material, is a non-abrasive mineral, is presented by micro-nano particles, and has more remarkable oral care effect as the particle size is smaller; the adhesive has strong binding force with pigment, can adsorb/inhibit bacteria and dental plaque fragments, and has remarkable anti-inflammatory and hemostatic effects; the tooth whitening agent can also permeate to the bottom of a tooth demineralization lesion, supplement mineral substances dissolved by dental plaque acid, restore the mineral substance density of teeth, seal dentinal tubules, realize bacteriostasis, remineralization restoration and desensitization, effectively prevent dental caries, and enhance translucence, glossiness and whiteness of dental enamel. In medicine carrying, the medicine carrying agent can be directly used for bone defect parts after being compounded with organic materials, not only can play a role in supporting bone defects, but also can inhibit the growth of bacteria and tumor cells through slow release of the medicine, and promote bone healing.
In the prior art, the application number is 202010962656.7, the publication date is 2021, 1 month and 1 day, the invention patent of the name of a bone tissue repair material, a preparation method and application thereof discloses that hydroxyapatite is firstly deposited on natural polymers to obtain hydroxyapatite-natural polymer nano composite particles, then the mixed particles are mixed with natural polymer solution, and then the bone tissue repair material is obtained through freeze drying. However, in the above technical scheme, the natural polymer surface is directly mineralized in situ, and because the preparation process is performed by using chemical reagents, the foreign body reaction is easy to occur when the mineralization process is applied to a human body, and in addition, the mineralization process is long in time and low in preparation efficiency.
In the prior art, the application number is 201410372692.2, the publication date is 2021, 1 and 1, and the invention patent entitled "method for improving the mechanical property of silk protein membrane by biomineralization" discloses that the silk protein membrane is soaked in CaCl 2 The silk fibroin film is pre-mineralized in the solution, and then the pre-mineralized silk fibroin film is soaked in the hydroxyapatite solution for mineralization. The preparation process can effectively avoid the generation of the hydroxyapatite byproducts and improve the mechanical property of the silk protein film, but the mineralized silk protein film can be applied to the body only through a complicated post-treatment process; in addition, the preparation process includes pre-mineralizing with calcium ion containing solution, adding mineralizing liquid of hydroxyapatite solution, and combining the hydroxyapatite in the mineralizing liquid with calcium ion adsorbed onto silk fibroinThe calcium-phosphorus ratio of the hydroxyapatite on the membrane is larger than that of normal bones of a human body, so that the hydroxyapatite has low compatibility with human tissues when used in the human body.
In view of the above, there is a need to design an improved preparation method and application of silk fibroin/hydroxyapatite composite micro-nano particle material to solve the above problems.
Disclosure of Invention
The invention aims to provide a preparation method and application of a silk fibroin/hydroxyapatite composite micro-nano particle material.
In order to achieve the aim of the invention, the invention provides a preparation method of a silk fibroin/hydroxyapatite composite micro-nano particle material, which comprises the following steps:
s1, freezing and solidifying a silk fibroin solution with a certain mass concentration to obtain a silk fibroin frozen body, and carrying out annealing treatment, thawing filtration and freeze drying to obtain silk fibroin nanoparticle powder;
s2, under a certain bath ratio, mineralizing the silk fibroin nanoparticle powder prepared in the step S1 with simulated body fluid to obtain the silk fibroin/hydroxyapatite composite micro-nanoparticle material.
Preferably, in step S2, before the mineralization reaction, auxiliary mineralization treatment is performed on the silk fibroin nanoparticle powder, specifically by the following steps: sequentially soaking the silk fibroin nanoparticle powder in a solution containing calcium ions and phosphate ions.
Preferably, in step S2, the bath ratio is (1:500) - (1:1500).
Preferably, in the step S2, the size of the silk fibroin/hydroxyapatite composite micro-nano particle material is 30-3000 nm; the mineralization reaction time is 2-7 days.
Preferably, in step S1, the mass concentration of the silk fibroin solution is 0.05-0.8 wt%; the annealing treatment temperature is-20-0 ℃ and the treatment time is 12-96 h.
Preferably, in step S1, the simulated body fluid is prepared 1 to 5 times as much as the composition of the body fluid of the human body.
Preferably, the calcium ion-containing solution is CaCl with the concentration of 100-200 mM 2 The solution containing phosphate ions is Na with the concentration of 100-200 mM 2 HPO 4 An aqueous solution; preferably, the time for soaking in the calcium ion-containing solution is 5-30 min, and the time for soaking in the phosphate ion-containing solution is 5-30 min.
Preferably, in the step S2, the mineralization reaction is carried out in an oscillating box at 37-40 ℃, and the reaction liquid is required to be subjected to centrifugal liquid exchange every 12-36 hours; preferably, the mineralization reaction is carried out at constant temperature.
Preferably, in step S2, after the silk fibroin nanoparticles are soaked in the calcium ion-containing solution and the phosphate ion-containing solution, the subsequent steps are performed after centrifugation and cleaning, the rotation speed of the centrifugation process is 4000-8000 r/min, the centrifugation time is 5-10 min, and the centrifugation temperature is 25-37 ℃.
In particular, the invention also provides application of the silk fibroin/hydroxyapatite composite micro-nano particle material, and the silk fibroin/hydroxyapatite composite micro-nano particle material can be used for dental restoration, oral care and medicine carrying.
The beneficial effects of the invention are as follows:
1. according to the preparation method of the silk fibroin/hydroxyapatite composite micro-nano particle material, silk fibroin nano particle powder is soaked in a solution containing calcium ions and phosphate ions in sequence to perform auxiliary mineralization reaction, nucleation sites are generated on the surfaces of the silk fibroin nano particles in the auxiliary mineralization reaction process, so that the growth of hydroxyapatite on the silk fibroin nano particles can be promoted, and meanwhile, the subsequent mineralization process is facilitated; then, the silk fibroin nanoparticle powder after auxiliary mineralization is soaked in simulated body fluid for mineralization reaction, so that hydroxyapatite crystals can be formed on the silk fibroin nanoparticle powder by self-assembly, and the silk fibroin/hydroxyapatite composite micro-nanoparticle material is finally prepared after freeze drying. By the method, the silk fibroin/hydroxyapatite composite micro-nano particle material which can be directly applied to the human body without post-treatment is prepared, and the problem that the composite micro-nano particle material in the prior art needs to be treated before being used in the human body is effectively solved.
2. According to the preparation method of the silk fibroin/hydroxyapatite composite micro-nano particle material, silk fibroin molecules are used as templates for assembling hydroxyapatite, so that the growth of hydroxyapatite crystals can be promoted, the advantages of good biocompatibility, bioactivity, biodegradability and the like of silk fibroin can be fully utilized, and the prepared silk fibroin/hydroxyapatite composite micro-nano particle material can induce bone repair; by carrying out auxiliary mineralization reaction and then mineralization reaction, calcium ions in mineralization liquid can be preferentially adsorbed to silk fibroin and combined with carboxyl groups on amino acid side chains of the silk fibroin in the auxiliary mineralization reaction process, and phosphate ions are further combined with calcium ions on the surface of the silk fibroin along with the introduction of phosphate ions to generate hydroxyapatite crystals, the process can enable the carboxyl groups and the calcium ions on the silk fibroin to be combined more fully, the calcium-phosphorus ratio of the hydroxyapatite in the composite nano particles is effectively improved, nucleation sites are generated by the auxiliary mineralization, the formation and crystallization of the hydroxyapatite are further accelerated, the reaction time of the mineralization reaction is effectively shortened, and meanwhile, the subsequent biomimetic mineralization process is facilitated; in addition, the surface structure of the silk fibroin can be changed through the pre-mineralization treatment, so that the morphology and the growth of the hydroxyapatite can be regulated and controlled, and the formation and the crystallization of the hydroxyapatite can be accelerated; by utilizing simulated body fluid to carry out mineralization treatment, the performance of the finally prepared silk fibroin/hydroxyapatite composite micro-nano particle material is more similar to that of normal bones of a human body, so that the silk fibroin/hydroxyapatite composite micro-nano particle material can be better compatible with human tissues, and the foreign body reaction of the material in the application process is effectively avoided. By the method, the silk fibroin/hydroxyapatite composite micro-nano particle material which has good biocompatibility and stability and can induce bone repair is prepared, and the problems that the composite material of the hydroxyapatite prepared in the prior art is not high in compatibility with a human body and long in mineralization process is consumed are solved.
Drawings
FIG. 1 is an SEM image of a silk fibroin/hydroxyapatite composite micro-nano particle material prepared in example 1 of the present invention;
FIG. 2 is an XRD pattern of the silk fibroin/hydroxyapatite composite micro-nano particle material prepared in example 1 of the present invention;
FIG. 3 is an IR chart of a silk fibroin/hydroxyapatite composite micro-nano particle material prepared in example 1 of the present invention;
FIG. 4 is a TG illustrating a silk fibroin/hydroxyapatite composite micro-nano particle material prepared in example 1 of the present invention;
FIG. 5 is a graph showing cytotoxicity detection data of silk fibroin/hydroxyapatite composite micro-nano particle material prepared in example 1 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in detail with reference to the accompanying drawings and specific embodiments.
It should be noted that, in order to avoid obscuring the present invention due to unnecessary details, only structures and/or processing steps closely related to aspects of the present invention are shown in the drawings, and other details not greatly related to the present invention are omitted.
In addition, it should be further noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Referring to fig. 1, the preparation method of the silk fibroin/hydroxyapatite composite micro-nano particle material provided by the invention comprises the following steps:
s1, preparing silk fibroin nanoparticle powder: freezing and solidifying a silk fibroin solution with a certain mass concentration to obtain a silk fibroin frozen body, and then carrying out annealing treatment, thawing, filtering and freeze drying to obtain silk fibroin nanoparticle powder;
s2, generating a silk fibroin/hydroxyapatite composite micro-nano particle material: under the condition of stirring, sequentially soaking the silk fibroin nano particles prepared in the step S1 in a solution containing calcium ions and phosphate ions for 5-30 min, and carrying out auxiliary mineralization reaction on silk fibroin nano particle powder by utilizing the pretreatment; then, under a certain bath ratio, mineralizing reaction is carried out on the pretreated silk fibroin nanoparticle powder and simulated body fluid, and the silk fibroin/hydroxyapatite composite micro-nanoparticle material is prepared.
Preferably, in the step S1, the mass concentration of the silk fibroin solution is 0.05-0.8 wt%, and when the concentration of the silk fibroin is in the above range, the obtained silk fibroin nanoparticles are uniform in size and complete in particle size; wherein the silk fibroin solution is prepared by the following steps:
immersing 100g of raw silk in 5L of sodium carbonate solution with the mass percentage of 0.1%, boiling at 100 ℃ for 30min, repeating the treatment twice, immersing the silk treated twice in 5L of sodium carbonate solution with the mass percentage of 0.05% for the third time, boiling at 100 ℃ for 30min, degumming the raw silk, and fully washing and drying to obtain pure silk fiber; next, the treated silk fibroin fibers were immersed in a molar ratio of 1:2:8, dissolving in an anhydrous calcium chloride/absolute ethyl alcohol/deionized water ternary solution at 72 ℃ for one hour; after cooling, pouring the dissolved solution into a dialysis bag with the molecular weight cutoff of 9-14 kDa, and dialyzing with deionized water to remove molecules with the molecular weight less than 9-14 kDa; filtering with gauze after dialysis to obtain silk fibroin solution; the resulting silk fibroin solution was then poured into a centrifuge tube, centrifuged to remove aggregates and bubbles, stored in a refrigerator at 4 ℃ for later use, and its concentration was determined by a dry mass method, and finally the silk fibroin solution was diluted to a desired concentration.
Preferably, in step S1, the annealing treatment is carried out at a temperature of-20 to 0 ℃ for a treatment time of 12 to 96 hours, and the annealing treatment aims at inducing assembly and structural transformation of the silk fibroin.
Preferably, in step S2, the simulated body fluid is prepared in 1 to 5 times the composition of the body fluid of the human body.
Preferably, in step S2, the calcium ion-containing solution is CaCl having a concentration of 100 to 200mM 2 Is an aqueous solution of (a); the solution containing phosphate radical ion is Na with the concentration of 100-200 mM 2 HPO 4 An aqueous solution.
Preferably, in the step S2, after the silk fibroin nano particles are soaked in the calcium ion-containing solution and the phosphate ion-containing solution, the silk fibroin nano particles are subjected to centrifugation and cleaning, and then the subsequent steps are carried out, wherein the rotating speed of the centrifugation process is 4000-8000 r/min, the centrifugation time is 5-10 min, and the temperature is 25-37 ℃.
Preferably, in the step S2, the size of the prepared silk fibroin/hydroxyapatite composite micro-nano particle material is 30-3000 nm.
Preferably, in the step S2, the mineralization reaction is carried out in an oscillating box at 37-40 ℃, the mineralization reaction process is carried out under the condition of constant temperature, and the reaction liquid is required to be subjected to centrifugal liquid exchange every 12-36 hours.
Preferably, in step S2, the bath ratio is (1:500) - (1:1500), and in the present invention, the bath ratio refers to the ratio of the mass of the particle powder to the volume of the simulated body fluid, and the unit is g/mL; when the bath ratio is in the above range, both the mineralization time and the mineralization degree can be kept at appropriate levels.
Preferably, in step S2, the mineralization time is 2 to 7 days.
In particular, the silk fibroin/hydroxyapatite composite micro-nano particle material prepared by the method can be used for dental restoration, oral care and drug loading.
The invention is further illustrated by the following examples:
example 1
S1, preparing silk fibroin nanoparticle powder:
the silk fibroin solution with the mass concentration of 0.4wt% is placed under the condition of liquid nitrogen for freezing and solidifying to obtain a silk fibroin frozen body, and then the silk fibroin frozen body is subjected to annealing treatment, thawing filtration and freeze drying to obtain silk fibroin nanoparticle powder; wherein, the annealing treatment is carried out in a refrigerator at the temperature of minus 4 ℃, the time of the annealing treatment is 48 hours, and the structural transformation of the silk fibroin can be induced in the annealing treatment process;
wherein the silk fibroin solution is prepared by the following steps: immersing 100g of raw silk in 5L of sodium carbonate solution with the mass percentage of 0.1%, boiling at 100 ℃ for 30min, repeating the treatment twice, immersing the silk treated twice in 5L of sodium carbonate solution with the mass percentage of 0.05% for the third time, boiling at 100 ℃ for 30min, degumming the raw silk, and fully washing and drying to obtain pure silk fiber; next, the treated silk fibroin fibers were immersed in a molar ratio of 1:2:8, dissolving in an anhydrous calcium chloride/absolute ethyl alcohol/deionized water ternary solution at 72 ℃ for one hour; after cooling, pouring the dissolved solution into a dialysis bag with the molecular weight cutoff of 9-14 kDa, and dialyzing with deionized water to remove molecules with the molecular weight less than 9-14 kDa; filtering with gauze after dialysis to obtain silk fibroin solution, pouring the silk fibroin solution into a centrifuge tube, centrifuging to remove aggregates and bubbles, storing in a refrigerator at 4 ℃ for standby, measuring the concentration by a dry mass method, and finally diluting the silk fibroin solution to 0.4wt%;
s2, generating silk fibroin/hydroxyapatite composite micro-nano particle material
Under the condition of stirring, the silk fibroin nano particles prepared in the step S1 are soaked in CaCl with the mass concentration of 150mM 2 Centrifuging the solution for 5 to 30 minutes, taking out the solution, and adding more CaCl by deionized water 2 The solution is removed, and then the washed silk fibroin nanoparticles are soaked in Na with the mass concentration of 150mM 2 HPO 4 Repeating the steps of cleaning and centrifuging in the solution for 5-30 min to obtain pretreated silk fibroin nano particles, wherein the rotating speed in the centrifuging process is 8000r/min, the centrifuging time is 5min, and the temperature is 25 ℃; after the above steps are completed, mineralizing reaction is carried out on the pretreated silk fibroin nano-particles and 1.5 times of simulated body fluid in a constant temperature shaking box at 37 ℃, and the bath ratio of the pretreated silk fibroin nano-particles to the simulated body fluid is 1:1000, the reaction time is 2 days, and the silk fibroin/hydroxyapatite composite micro-nano particle material is prepared after the reaction is finished and freeze-dried. Silk fibroin/hydroxyapatite composite micro-nano prepared in example 1As can be seen from the figure 2, the XRD patterns and the IR patterns of the rice particle material are shown in the figure 2, after the self-assembly on the silk fibroin nano particles forms hydroxyapatite crystals, characteristic diffraction peaks of the silk fibroin nano particles are reserved in the silk fibroin/hydroxyapatite composite micro-nano particle material, and meanwhile, characteristic peaks of the hydroxyapatite are also generated, so that the result shows that the silk fibroin/hydroxyapatite composite micro-nano particle material is successfully prepared; as can be seen from fig. 3, before and after the self-assembly of the hydroxyapatite crystals on the silk fibroin nanoparticles, the peak of the spectrum did not change greatly, indicating that the structure of the silk fibroin nanoparticles was not changed after the self-assembly of the hydroxyapatite crystals on the silk fibroin nanoparticles. Fig. 4 is a TG diagram of a silk fibroin/hydroxyapatite composite micro-nanoparticle material, and the result shows that after self-assembling the formed hydroxyapatite on silk fibroin, the thermal stability and the oxidation stability of the nanoparticle can be improved.
Example 2
Example 2 differs from example 1 only in that: the silk fibroin nanoparticle powder is mineralized in the simulated body fluid without auxiliary mineralization reaction to form silk fibroin/hydroxyapatite composite micro-nanoparticles, and other steps are basically the same as those of the embodiment 1, and are not repeated here. The mineralization time in example 2 was 5 days longer than 2 days in example 1, and the mineralization degree, uniformity and stability of hydroxyapatite in the obtained silk fibroin/hydroxyapatite composite micro-nano particles were not as good as those of the silk fibroin/hydroxyapatite composite micro-nano particles prepared in example 1.
Comparative example 1
Comparative example 1 differs from example 1 only in that: the steps other than self-assembling hydroxyapatite on silk fibroin nanoparticles are substantially the same as in example 1, and will not be described again here.
In order to compare the toxicity of the silk fibroin/hydroxyapatite composite micro-nano particle material prepared in the example 1 and the comparative example 1, the composite micro-nano particle is utilized to perform a non-toxicity test on bone cells under the same condition, and in addition, the influence of the concentration of the composite micro-nano particle material on cytotoxicity is also considered in the test process. The results of the tests are shown in FIG. 5, in which the difference ratio of absorbance values is used to reflect the viability of bone cells, and it can be seen from the graph that the cell activity in both tests is 80% or more in the presence of the nanoparticles of example 1 and comparative example 1, indicating that neither of the nanoparticles prepared in example 1 and comparative example 1 has cytotoxicity; with the increase of the concentration, the cell activity is gradually improved, and when the concentration is 0.1mg/mL and 0.5mg/mL, the cell activity of the composite nanoparticle is slightly higher than that of the pure silk fibroin nanoparticle, but when the concentration reaches 1mg/mL, the cell activity of the composite nanoparticle is obviously higher than that of the pure silk fibroin nanoparticle, and the result shows that: compared with pure silk fibroin nano particles, the silk fibroin/hydroxyapatite composite micro-nano particles can promote the growth of osteoblasts, and with the increase of the concentration, the silk fibroin/hydroxyapatite composite micro-nano particles are more beneficial to the growth of the osteoblasts.
Comparative example 2
Comparative example 2 differs from example 1 only in that: comparative example 2 in which only silk fibroin nanoparticle powder was immersed in CaCl 2 The auxiliary mineralization reaction was performed in the solution, and other steps are substantially the same as in example 1, and will not be described here. In the auxiliary mineralization process, ca 2+ The ions are firstly adsorbed on the silk fibroin, and then Ca in the body fluid 2+ And HPO 4 2- Further self-assembling on the silk fibroin to form the hydroxyapatite, wherein the calcium-phosphorus ratio of the silk fibroin/hydroxyapatite composite micro-nano particle material prepared by the process is lower than that of the composite micro-nano particle material prepared in the example 1. Therefore, the composite micro-nano particles prepared in comparative example 2 have lower bone cell activity than the composite micro-nano particles prepared in example 1.
In summary, according to the preparation method of the silk fibroin/hydroxyapatite composite micro-nano particle material provided by the invention, silk fibroin nano particle powder is sequentially soaked in a solution containing calcium ions and phosphate ions to carry out auxiliary mineralization reaction, nucleation sites are generated on the surfaces of the silk fibroin nano particles in the auxiliary mineralization reaction process, so that the formation and crystallization of hydroxyapatite crystals can be accelerated, the reaction time of mineralization reaction is effectively shortened, and meanwhile, the subsequent mineralization process is facilitated; and then, mineralizing the mineralized silk fibroin nanoparticle powder with simulated body fluid to perform a mineralization reaction, so that hydroxyapatite can be formed on the silk fibroin nanoparticle powder by self-assembly, and finally the silk fibroin/hydroxyapatite composite micro-nanoparticle material is prepared after freeze drying. By the method, the silk fibroin/hydroxyapatite composite micro-nano particle material which has good biocompatibility and stability and can induce bone repair is prepared.
The above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention.
Claims (6)
1. The preparation method of the silk fibroin/hydroxyapatite composite micro-nano particle material is characterized by comprising the following steps of:
s1, freezing and solidifying a silk fibroin solution with a certain mass concentration to obtain a silk fibroin frozen body, and carrying out annealing treatment, thawing filtration and freeze drying to obtain silk fibroin nanoparticle powder, wherein the mass concentration of the silk fibroin solution is 0.05-0.8wt%; the temperature of the annealing treatment is-20-0 ℃ and the treatment time is 12-96 h;
s2, sequentially soaking the silk fibroin nanoparticle powder into a solution containing calcium ions and phosphate ions, and performing auxiliary mineralization treatment, wherein the solution containing the calcium ions is CaCl with the concentration of 100-200 mM 2 The solution containing phosphate ions is Na with the concentration of 100-200 mM 2 HPO 4 The time for soaking the silk fibroin nanoparticle powder in the calcium ion-containing solution is 5-30 min, and the time for soaking the silk fibroin nanoparticle powder in the phosphate ion-containing solution is 5-30 min; and then mineralizing the silk fibroin nanoparticle powder prepared in the step S1 with simulated body fluid under a certain bath ratio, and freeze-drying to obtain the silk fibroin nanoparticle powder with the size of 30-300The silk fibroin/hydroxyapatite composite micro-nano particle material with the wavelength of 0nm is prepared from the simulated body fluid according to 1-3 times of the components of the body fluid of a human body.
2. The method for preparing silk fibroin/hydroxyapatite composite micro-nano particle material according to claim 1, wherein in step S2, the bath ratio is (1:500) - (1:1500).
3. The method for preparing silk fibroin/hydroxyapatite composite micro-nano particle material according to claim 1, wherein in step S2, the mineralization reaction time is 2-7 days.
4. The method for preparing the silk fibroin/hydroxyapatite composite micro-nano particle material according to claim 1, wherein in the step S2, the mineralization reaction is performed in an oscillating box at 37-40 ℃, and the reaction liquid is required to be subjected to centrifugal liquid exchange every 12-36 hours; the mineralization reaction is carried out under the condition of constant temperature.
5. The method for preparing the silk fibroin/hydroxyapatite composite micro-nano particle material according to claim 4, wherein in the step S2, the silk fibroin nano particles are soaked in the calcium ion-containing solution and the phosphate ion-containing solution, and then the subsequent steps are carried out after centrifugation and cleaning, the rotational speed of the centrifugation process is 4000-8000 r/min, the centrifugation time is 5-10 min, and the centrifugation temperature is 25-37 ℃.
6. The use of a silk fibroin/hydroxyapatite composite micro-nano particle material according to any one of claims 1 to 5, wherein the silk fibroin/hydroxyapatite composite micro-nano particle material can be used for preparing dental restoration, oral care and drug-carrying materials.
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