CN119770747B - A method for preparing a polymer filler containing silk fibroin particles - Google Patents
A method for preparing a polymer filler containing silk fibroin particles Download PDFInfo
- Publication number
- CN119770747B CN119770747B CN202510053643.0A CN202510053643A CN119770747B CN 119770747 B CN119770747 B CN 119770747B CN 202510053643 A CN202510053643 A CN 202510053643A CN 119770747 B CN119770747 B CN 119770747B
- Authority
- CN
- China
- Prior art keywords
- silk fibroin
- solution
- particles
- silk
- sheep
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Processes Of Treating Macromolecular Substances (AREA)
- Materials For Medical Uses (AREA)
- Compositions Of Macromolecular Compounds (AREA)
- Cosmetics (AREA)
Abstract
The invention discloses a preparation method of a polymer filler containing silk fibroin particles, and relates to the technical field of polymer fillers. The invention adds silk fibroin gel particles and concentrated silk fibroin solution simultaneously, and self-enhanced assembly of the concentrated silk fibroin and silk fibroin gel improves the mechanical property of the silk fibroin particles. The invention also adds sheep collagen fiber and sulfhydrylation hyaluronic acid, which can prolong degradation time and provide bracket space for tissue regeneration. Meanwhile, hydrogen peroxide is used for crosslinking, and the concentration of the hydrogen peroxide is controlled to be 5-5.5mmol/L, so that the stability of the material is improved, the biodegradation rate of the material is reduced, and the degradation time is further prolonged.
Description
Technical Field
The invention relates to the technical field of polymer fillers, in particular to a preparation method of a polymer filler containing silk fibroin particles.
Background
Silk fibroin is a natural and old protein material, can be extracted from artificially cultured cocoons, has good biocompatibility, adjustable biodegradability and non-immunogenicity, can be used as a suture material in the biomedical field, and is a biological material which can be used in clinic. Silk fibroin materials are easy to process, but are thermodynamically unstable, and can be regulated and controlled by physical and chemical methods, so that the content of beta-sheet structures in silk fibroin is promoted to be increased, and the silk fibroin is gradually converted into a thermodynamically stable structure. The structural adjustability and the rapid transformation capability enable the silk fibroin material to be flexibly adjusted and controlled according to the actual requirements on the appearance, mechanical property, biodegradability and the like of the material when the silk fibroin material is used as a tissue engineering material.
On the other hand, silk fibroin is rich in 18 amino acids, has no toxicity to tissues and organisms, and degradation products of silk fibroin cannot cause adverse reactions. Meanwhile, more dressing products containing silk fibroin components are sold on the market, and the biological safety of the silk fibroin is proved. Thus, silk fibroin can be used as a raw material for implant filling. Different tissue sites have different requirements on the size of the filling particles, and the silk fibroin has good morphological processability, so that the silk fibroin particles are compounded with other polymers such as collagen, hyaluronic acid and the like.
In order to solve the above problems, the present invention provides a method for preparing a polymer filler containing silk fibroin particles.
Disclosure of Invention
The invention aims to provide a preparation method of a polymer filler containing silk fibroin particles, which aims to solve the problems in the prior art.
In order to achieve the above purpose, the present invention provides the following technical solutions:
a method of preparing a polymer filler comprising silk fibroin particles, comprising the steps of:
Dissolving degummed silk with lithium bromide solution to obtain dissolved silk, cooling the dissolved silk, dialyzing, and taking out to obtain silk solution;
step two, taking a silk fibroin solution, filtering, centrifuging, concentrating at 60-65 ℃, and adding water for injection for dilution to obtain a diluted silk fibroin solution;
Thirdly, placing the diluted silk fibroin solution at 60-65 ℃ until silk fibroin gel is formed;
Step four, taking silk fibroin gel, granulating, adding concentrated silk fibroin solution into the granulated silk fibroin gel particles, uniformly mixing, freezing at-40 ℃ to-80 ℃ for 24-48 hours, freeze-drying, absorbing moisture at 25-30 ℃ for 1-3d, and crushing to obtain silk fibroin freeze-dried particles;
Mixing silk fibroin freeze-dried particles, sulfhydryl hyaluronic acid and deionized water uniformly, regulating the pH value of the system to 8-8.5 by using a sodium hydroxide solution, adding sulfhydryl sheep collagen fiber particles, mixing uniformly at 0-2 ℃, adding hydrogen peroxide, reacting for 20-30min at 35-40 ℃, dialyzing, freeze-drying to obtain particles A, taking the particles A and sodium hyaluronate gel, and mixing uniformly to obtain the polymer filler containing silk fibroin particles.
More preferably, in the first step, the bath ratio of degummed silk to lithium bromide is 1 (4-10).
More preferably, in the second step, the concentration of the diluted silk fibroin solution is 2-6wt%.
More preferably, in the fourth step, the mass ratio of the granulated silk fibroin gel particles to the silk fibroin in the concentrated silk fibroin solution is 1 (0.2-2).
More preferably, in the second step, the centrifugation condition is centrifugation at 9000rpm for 20-30min.
More optimally, the preparation method of the sulfhydrylation sheep collagen fiber particles comprises the steps of uniformly mixing sheep collagen fibers and acetic acid to obtain a sheep collagen fiber solution, cooling to 2-3 ℃, adding N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, uniformly mixing, reacting for 30-40min, then dropwise adding a cysteamine hydrochloride solution, adding a 2- (N-morpholino) ethanesulfonic acid solution, regulating the pH value of the solution to 5.5-6, stirring for 4-5h, reacting for 10-14h at 4-5 ℃, dialyzing to obtain the sulfhydrylation sheep collagen fiber solution, and freeze-drying to obtain the sulfhydrylation sheep collagen fiber particles.
More optimally, the preparation method of the sheep collagen fibers comprises the steps of dissolving sheep collagen powder in acetic acid, dialyzing to neutrality to obtain sheep collagen solution, adding phosphate buffer solution, uniformly mixing, centrifuging, then re-suspending the precipitate in the phosphate buffer solution, centrifuging, and drying to obtain the sheep collagen fibers.
More optimally, the preparation method of the sheep collagen powder comprises the steps of taking sheep skin, smashing, washing, degreasing by using n-butanol, centrifuging, washing, extracting collagen by using acetic acid solution containing pepsin, regulating pH value to be neutral, inactivating enzyme, dialyzing, and freeze-drying to obtain the sheep collagen powder.
More preferably, the concentration of the hydrogen peroxide is 5-5.5mmol/L.
Compared with the prior art, the invention has the beneficial effects that:
1. When the silk fibroin particles are compounded with substances such as collagen, hyaluronic acid and the like and used as a filler of each tissue, the silk fibroin particles can have the advantages of various materials. At this time, the polymer filler can play a role in supporting and filling, and the silk fibroin particles are gradually released along with the degradation process, so that the effect of stimulating tissue regeneration is played again.
2. The invention adds silk fibroin gel particles and concentrated silk fibroin solution simultaneously, and self-enhanced assembly of the concentrated silk fibroin and silk fibroin gel improves the mechanical property of the silk fibroin particles.
3. The sheep collagen fibers and the thiolated hyaluronic acid are added, so that the degradation time can be prolonged, and a bracket space is provided for tissue regeneration. The invention also uses N-hydroxysuccinimide, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 2- (N-morpholino) ethane sulfonic acid to carry out sulfhydrylation modification on sheep collagen fibers, so that the sheep collagen fibers contain sulfhydryl groups, sulfhydryl groups exist on sulfhydryl hyaluronic acid and silk fibroin freeze-dried particles, the three have good compatibility, the degradation time is better prolonged, and simultaneously, the invention also uses hydrogen peroxide to crosslink the sheep collagen fibers, and the stability of the material is improved, the biodegradation rate of the sheep collagen fibers is reduced, and the degradation time is further prolonged by controlling the concentration of the hydrogen peroxide to be 5-5.5 mmol/L.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The source and model of the substances related to the invention are not particularly limited, and exemplary substances include sulfhydryl hyaluronic acid, SH-HA-372, 6A raw silk, pepsin, sodium hyaluronate gel, 20200701, shandong Baolijia biosciences, inc. of the Shandong standard technical service, inc. of the Shandong Baolijia biosciences, inc. of the Shandong Lijia biosciences, and degumming raw silk by the raw silk.
Example 1A process for the preparation of a polymer filler containing silk fibroin particles comprising the steps of:
step one, preparing silk fibroin freeze-dried particles:
dissolving degummed silk with 9.3mol/L lithium bromide solution at 60 ℃, wherein the bath ratio of degummed silk to lithium bromide is 1:5, and obtaining dissolved silk;
cooling the dissolved silk fibroin, putting the silk fibroin into a dialysis bag with a cut-off molecular weight of 14kDa, dialyzing with purified water for three days, and taking out the silk fibroin solution to obtain a silk fibroin solution;
Taking silk fibroin solution, filtering, centrifuging for 25min at 4 ℃ and 9000rpm to obtain silk fibroin solution with concentration of 5wt%, concentrating at 62 ℃ to 17wt%, adding water for injection into the concentrated silk fibroin solution, and diluting to 4wt% to obtain diluted silk fibroin solution;
placing the diluted silk fibroin solution at 60 ℃ again until silk fibroin gel is formed;
Granulating silk fibroin gel through a screen, adding a concentrated silk fibroin solution into the granulated silk fibroin gel particles, uniformly mixing the silk fibroin gel particles and the concentrated silk fibroin solution at a mass ratio of 1:1, freezing at-60 ℃ for 36h, freeze-drying, dehydrating at room temperature for 2d, and crushing to obtain silk fibroin freeze-dried particles;
step two, preparing a polymer filler containing silk fibroin particles:
Mixing 18g of silk fibroin freeze-dried particles, 5g of sulfhydryl hyaluronic acid and 1000mL of deionized water uniformly, regulating the pH value of a system to 8 by using a 1mol/L sodium hydroxide solution, mixing uniformly at 1 ℃, adding 5mmol/L hydrogen peroxide, reacting for 25min at 38 ℃, dialyzing, freeze-drying to obtain particles A, taking 16 parts of particles A and sodium hyaluronate gel, mixing uniformly, and obtaining the polymer filler containing silk fibroin particles.
Example 2A process for the preparation of a polymer filler containing silk fibroin particles comprising the steps of:
step one, preparing silk fibroin freeze-dried particles:
dissolving degummed silk with 9.3mol/L lithium bromide solution at 60 ℃, wherein the bath ratio of degummed silk to lithium bromide is 1:5, and obtaining dissolved silk;
cooling the dissolved silk fibroin, putting the silk fibroin into a dialysis bag with a cut-off molecular weight of 14kDa, dialyzing with purified water for three days, and taking out the silk fibroin solution to obtain a silk fibroin solution;
Taking silk fibroin solution, filtering, centrifuging for 25min at 4 ℃ and 9000rpm to obtain silk fibroin solution with concentration of 5wt%, concentrating at 62 ℃ to 17wt%, adding water for injection into the concentrated silk fibroin solution, and diluting to 4wt% to obtain diluted silk fibroin solution;
placing the diluted silk fibroin solution at 60 ℃ again until silk fibroin gel is formed;
Granulating silk fibroin gel through a screen, adding a concentrated silk fibroin solution into the granulated silk fibroin gel particles, uniformly mixing the silk fibroin gel particles and the concentrated silk fibroin solution at a mass ratio of 1:1, freezing at-60 ℃ for 36h, freeze-drying, dehydrating at room temperature for 2d, and crushing to obtain silk fibroin freeze-dried particles;
step two, preparing sheep collagen fibers:
Crushing and washing sheep skin, degreasing with 10% n-butanol, centrifuging, washing with water, extracting collagen with 0.5M acetic acid solution containing 1g/L pepsin, adjusting pH to neutrality, inactivating enzyme, dialyzing, and lyophilizing to obtain sheep collagen powder;
Preparation of sheep collagen fibers:
Dissolving sheep collagen powder in 0.5M acetic acid, dialyzing to neutrality to obtain sheep collagen solution of 3.5mg/mL, adding 20mM phosphate buffer solution, mixing uniformly, centrifuging, suspending the precipitate in phosphate buffer solution at concentration of 3mg/mL, centrifuging, and drying to obtain sheep collagen fiber;
Step three: preparation of sulfhydrylation sheep collagen fiber:
Uniformly mixing 3g of sheep collagen fibers, 50mL and 0.5M acetic acid to obtain a sheep collagen fiber solution, cooling to 2 ℃, adding 0.2g of N-hydroxysuccinimide and 0.4g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, uniformly mixing, reacting for 35min, then dropwise adding 50mg/mL of cysteamine hydrochloride solution to ensure that the concentration of cysteamine hydrochloride in the final solution is 55mmol/L, adding 2- (N-morpholino) ethanesulfonic acid solution, adjusting the pH value of the solution to 5.5, stirring for 4.5h, reacting for 12h at 4 ℃, dialyzing to obtain a sulfhydrylated sheep collagen fiber solution, and lyophilizing to obtain sulfhydrylated sheep collagen fiber particles;
step four, preparing a polymer filler containing silk fibroin particles:
Mixing 18g of silk fibroin freeze-dried particles, 5g of sulfhydryl hyaluronic acid and 1000mL of deionized water uniformly, regulating the pH value of a system to 8 by using a 1mol/L sodium hydroxide solution, adding 3g of sulfhydryl sheep collagen fiber particles, mixing uniformly at 1 ℃, adding 5mmol/L hydrogen peroxide, reacting at 38 ℃ for 25min, dialyzing, freeze-drying to obtain particles A, taking 16 parts of particles A and sodium hyaluronate gel, and mixing uniformly to obtain the polymer filler containing silk fibroin particles.
Example 3A process for the preparation of a polymer filler containing silk fibroin particles comprising the steps of:
step one, preparing silk fibroin freeze-dried particles:
Dissolving degummed silk with 9.3mol/L lithium bromide solution at 60 ℃, wherein the bath ratio of degummed silk to lithium bromide is 1:4, and obtaining dissolved silk;
cooling the dissolved silk fibroin, putting the silk fibroin into a dialysis bag with a cut-off molecular weight of 14kDa, dialyzing with purified water for three days, and taking out the silk fibroin solution to obtain a silk fibroin solution;
taking silk fibroin solution, filtering, centrifuging for 20min at 5 ℃ and 9000rpm to obtain silk fibroin solution with concentration of 3wt%, concentrating at 60 ℃ to 15wt%, adding water for injection into the concentrated silk fibroin solution, and diluting to 2wt% to obtain diluted silk fibroin solution;
placing the diluted silk fibroin solution at 60 ℃ again until silk fibroin gel is formed;
Granulating silk fibroin gel through a screen, adding a concentrated silk fibroin solution into the granulated silk fibroin gel particles, uniformly mixing the granulated silk fibroin gel particles and the concentrated silk fibroin solution at a mass ratio of 1:0.5, freezing for 24 hours at-40 ℃, freeze-drying, dehydrating at room temperature for 1d, and crushing to obtain silk fibroin freeze-dried particles;
step two, preparing sheep collagen fibers:
Crushing and washing sheep skin, degreasing with 10% n-butanol, centrifuging, washing with water, extracting collagen with 0.5M acetic acid solution containing 1g/L pepsin, adjusting pH to neutrality, inactivating enzyme, dialyzing, and lyophilizing to obtain sheep collagen powder;
Dissolving sheep collagen powder in 0.5M acetic acid, dialyzing to neutrality to obtain sheep collagen solution of 3.5mg/mL, adding 20mM phosphate buffer solution, mixing uniformly, centrifuging, suspending the precipitate in phosphate buffer solution at concentration of 3mg/mL, centrifuging, and drying to obtain sheep collagen fiber;
Step three: preparation of sulfhydrylation sheep collagen fiber:
Uniformly mixing 3g of sheep collagen fibers, 50mL and 0.5M acetic acid to obtain a sheep collagen fiber solution, cooling to 2 ℃, adding 0.2g of N-hydroxysuccinimide and 0.4g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, uniformly mixing, reacting for 30min, then dropwise adding 50mg/mL of cysteamine hydrochloride solution to ensure that the concentration of cysteamine hydrochloride in the final solution is 55mmol/L, adding 2- (N-morpholino) ethanesulfonic acid solution, adjusting the pH value of the solution to 5.5, stirring for 4h, reacting for 10h at 4 ℃, dialyzing to obtain a sulfhydrylated sheep collagen fiber solution, and freeze-drying to obtain sulfhydrylated sheep collagen fiber particles;
step four, preparing a polymer filler containing silk fibroin particles:
Mixing 18g of silk fibroin freeze-dried particles, 5g of sulfhydryl hyaluronic acid and 1000mL of deionized water uniformly, regulating the pH value of a system to 8 by using a 1mol/L sodium hydroxide solution, adding 3g of sulfhydryl sheep collagen fiber particles, mixing uniformly at 0 ℃, adding 5mmol/L hydrogen peroxide, reacting for 20min at 35 ℃, dialyzing, and freeze-drying to obtain particles A, taking 16 parts of particles A and sodium hyaluronate gel, and mixing uniformly to obtain the polymer filler containing silk fibroin particles.
Example 4A process for the preparation of a polymer filler containing silk fibroin particles comprising the steps of:
step one, preparing silk fibroin freeze-dried particles:
dissolving degummed silk with 9.3mol/L lithium bromide solution at 60 ℃, wherein the bath ratio of degummed silk to lithium bromide is 1:10, and obtaining dissolved silk;
cooling the dissolved silk fibroin, putting the silk fibroin into a dialysis bag with a cut-off molecular weight of 14kDa, dialyzing with purified water for three days, and taking out the silk fibroin solution to obtain a silk fibroin solution;
Taking silk fibroin solution, filtering, centrifuging for 30min at 5 ℃ and 9000rpm to obtain silk fibroin solution with the concentration of 6wt%, concentrating at 65 ℃ to 20wt%, adding water for injection into the concentrated silk fibroin solution, and diluting to 6wt% to obtain diluted silk fibroin solution;
placing the diluted silk fibroin solution at 60 ℃ again until silk fibroin gel is formed;
granulating silk fibroin gel through a screen, adding a concentrated silk fibroin solution into the granulated silk fibroin gel particles, uniformly mixing the silk fibroin gel particles and the concentrated silk fibroin solution at a mass ratio of 1:2, freezing at-80 ℃ for 48 hours, freeze-drying, dehydrating at room temperature for 3d, and crushing to obtain silk fibroin freeze-dried particles;
step two, preparing sheep collagen fibers:
Crushing and washing sheep skin, degreasing with 10% n-butanol, centrifuging, washing with water, extracting collagen with 0.5M acetic acid solution containing 1g/L pepsin, adjusting pH to neutrality, inactivating enzyme, dialyzing, and lyophilizing to obtain sheep collagen powder;
Preparation of sheep collagen fibers:
Dissolving sheep collagen powder in 0.5M acetic acid, dialyzing to neutrality to obtain sheep collagen solution of 3.5mg/mL, adding 20mM phosphate buffer solution, mixing uniformly, centrifuging, suspending the precipitate in phosphate buffer solution at concentration of 3mg/mL, centrifuging, and drying to obtain sheep collagen fiber;
Step three: preparation of sulfhydrylation sheep collagen fiber:
uniformly mixing 3g of sheep collagen fibers, 50mL and 0.5M acetic acid to obtain a sheep collagen fiber solution, cooling to 3 ℃, adding 0.2g of N-hydroxysuccinimide and 0.4g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, uniformly mixing, reacting for 40min, then dropwise adding 50mg/mL of cysteamine hydrochloride solution to ensure that the concentration of cysteamine hydrochloride in the final solution is 55mmol/L, adding 2- (N-morpholino) ethanesulfonic acid solution, regulating the pH value of the solution to 6, stirring for 5h, reacting for 14h at5 ℃, dialyzing to obtain a thiolated sheep collagen fiber solution, and freeze-drying to obtain thiolated sheep collagen fiber particles;
step four, preparing a polymer filler containing silk fibroin particles:
Mixing 18g of silk fibroin freeze-dried particles, 5g of sulfhydryl hyaluronic acid and 1000mL of deionized water uniformly, regulating the pH value of a system to 8 by using a 1mol/L sodium hydroxide solution, adding 3g of sulfhydryl sheep collagen fiber particles, mixing uniformly at 2 ℃, adding 5mmol/L hydrogen peroxide, reacting for 30min at 40 ℃, dialyzing, and freeze-drying to obtain particles A, taking 16 parts of particles A and sodium hyaluronate gel, and mixing uniformly to obtain the polymer filler containing silk fibroin particles.
Comparative example 1 sheep collagen fibers were not thiol modified and the rest was the same as in example 2:
step one, preparing silk fibroin freeze-dried particles:
dissolving degummed silk with 9.3mol/L lithium bromide solution at 60 ℃, wherein the bath ratio of degummed silk to lithium bromide is 1:5, and obtaining dissolved silk;
cooling the dissolved silk fibroin, putting the silk fibroin into a dialysis bag with a cut-off molecular weight of 14kDa, dialyzing with purified water for three days, and taking out the silk fibroin solution to obtain a silk fibroin solution;
Taking silk fibroin solution, filtering, centrifuging for 25min at 4 ℃ and 9000rpm to obtain silk fibroin solution with concentration of 5wt%, concentrating at 62 ℃ to 17wt%, adding water for injection into the concentrated silk fibroin solution, and diluting to 4wt% to obtain diluted silk fibroin solution;
placing the diluted silk fibroin solution at 60 ℃ again until silk fibroin gel is formed;
Granulating silk fibroin gel through a screen, adding a concentrated silk fibroin solution into the granulated silk fibroin gel particles, uniformly mixing the silk fibroin gel particles and the concentrated silk fibroin solution at a mass ratio of 1:1, freezing at-60 ℃ for 36h, freeze-drying, dehydrating at room temperature for 2d, and crushing to obtain silk fibroin freeze-dried particles;
step two, preparing sheep collagen fibers:
Crushing and washing sheep skin, degreasing with 10% n-butanol, centrifuging, washing with water, extracting collagen with 0.5M acetic acid solution containing 1g/L pepsin, adjusting pH to neutrality, inactivating enzyme, dialyzing, and lyophilizing to obtain sheep collagen powder;
Dissolving sheep collagen powder in 0.5M acetic acid, dialyzing to neutrality to obtain sheep collagen solution of 3.5mg/mL, adding 20mM phosphate buffer solution, mixing uniformly, centrifuging, suspending the precipitate in phosphate buffer solution at concentration of 3mg/mL, centrifuging, and drying to obtain sheep collagen fiber;
step three, preparing sheep collagen fiber particles:
uniformly mixing 3g of sheep collagen fibers, 50mL of acetic acid with the concentration of 0.5M to obtain sheep collagen fiber solution, and freeze-drying to obtain sheep collagen fiber particles;
step four, preparing a polymer filler containing silk fibroin particles:
mixing 18g of silk fibroin freeze-dried particles, 5g of sulfhydryl hyaluronic acid and 1000mL of deionized water uniformly, regulating the pH value of a system to 8 by using a 1mol/L sodium hydroxide solution, adding 3g of sheep collagen fiber particles, mixing uniformly at 1 ℃, adding 5mmol/L hydrogen peroxide, reacting at 38 ℃ for 25min, dialyzing, and freeze-drying to obtain particles A, taking 16 parts of particles A and sodium hyaluronate gel, and mixing uniformly to obtain the polymer filler containing silk fibroin particles.
Comparative example 2 thiol-modified hyaluronic acid was not added, and the rest was the same as example 2:
step one, preparing silk fibroin freeze-dried particles:
dissolving degummed silk with 9.3mol/L lithium bromide solution at 60 ℃, wherein the bath ratio of degummed silk to lithium bromide is 1:5, and obtaining dissolved silk;
cooling the dissolved silk fibroin, putting the silk fibroin into a dialysis bag with a cut-off molecular weight of 14kDa, dialyzing with purified water for three days, and taking out the silk fibroin solution to obtain a silk fibroin solution;
Taking silk fibroin solution, filtering, centrifuging for 25min at 4 ℃ and 9000rpm to obtain silk fibroin solution with concentration of 5wt%, concentrating at 62 ℃ to 17wt%, adding water for injection into the concentrated silk fibroin solution, and diluting to 4wt% to obtain diluted silk fibroin solution;
placing the diluted silk fibroin solution at 60 ℃ again until silk fibroin gel is formed;
Granulating silk fibroin gel through a screen, adding a concentrated silk fibroin solution into the granulated silk fibroin gel particles, uniformly mixing the silk fibroin gel particles and the concentrated silk fibroin solution at a mass ratio of 1:1, freezing at-60 ℃ for 36h, freeze-drying, dehydrating at room temperature for 2d, and crushing to obtain silk fibroin freeze-dried particles;
step two, preparing sheep collagen fibers:
Crushing and washing sheep skin, degreasing with 10% n-butanol, centrifuging, washing with water, extracting collagen with 0.5M acetic acid solution containing 1g/L pepsin, adjusting pH to neutrality, inactivating enzyme, dialyzing, and lyophilizing to obtain sheep collagen powder;
Preparation of sheep collagen fibers:
Dissolving sheep collagen powder in 0.5M acetic acid, dialyzing to neutrality to obtain sheep collagen solution of 3.5mg/mL, adding 20mM phosphate buffer solution, mixing uniformly, centrifuging, suspending the precipitate in phosphate buffer solution at concentration of 3mg/mL, centrifuging, and drying to obtain sheep collagen fiber;
Step three: preparation of sulfhydrylation sheep collagen fiber:
Uniformly mixing 3g of sheep collagen fibers, 50mL and 0.5M acetic acid to obtain a sheep collagen fiber solution, cooling to 2 ℃, adding 0.2g of N-hydroxysuccinimide and 0.4g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, uniformly mixing, reacting for 35min, then dropwise adding 50mg/mL of cysteamine hydrochloride solution to ensure that the concentration of cysteamine hydrochloride in the final solution is 55mmol/L, adding 2- (N-morpholino) ethanesulfonic acid solution, adjusting the pH value of the solution to 5.5, stirring for 4.5h, reacting for 12h at 4 ℃, dialyzing to obtain a sulfhydrylated sheep collagen fiber solution, and lyophilizing to obtain sulfhydrylated sheep collagen fiber particles;
step four, preparing a polymer filler containing silk fibroin particles:
Mixing 18g of silk fibroin freeze-dried particles and 1000mL of deionized water uniformly, regulating the pH value of a system to 8 by using a 1mol/L sodium hydroxide solution, adding 3g of sulfhydrylation sheep collagen fiber particles, uniformly mixing at 1 ℃, adding 5mmol/L hydrogen peroxide, reacting at 38 ℃ for 25min, dialyzing, and freeze-drying to obtain particles A, taking 16 parts of particles A and sodium hyaluronate gel, and uniformly mixing to obtain the polymer filler containing silk fibroin particles.
Comparative example 3 in the fourth step, the concentration of hydrogen peroxide was 3mmol/L, and the rest was the same as in example 2.
Experiment:
Experiment 1. In vitro degradation Performance test, the polymer filler containing silk fibroin particles obtained in examples 1-4 and comparative examples 1-3 is weighed, the initial weight is recorded as m0, the polymer filler is transferred and soaked into a phosphate buffer solution containing 20U/mL collagenase, the phosphate buffer solution is placed into a gas bath oscillation box with the temperature of 37 ℃ and the oscillation speed of 150rpm for experiment, samples are taken out at intervals, after freeze drying, the weight is weighed as m1, new degradation liquid is replaced until the condition (m 0-m 1)/m 0 is more than 0.99, the complete degradation is considered, and the complete degradation time is recorded.
Experiment 2. Injectability test Polymer fillers containing silk fibroin particles obtained in examples 1-4 and comparative examples 1-3 were filled into 1mL syringes, and 27G injection needles were mounted to simulate actual use. The pushing rod is pushed at a constant pushing speed of 30mm/min, the pushing displacement is 20mm, the sample in the syringe is pushed out through the needle, and the compression load of the injectable gel ranges from 8N to 16N within a certain compression displacement.
| Degradation time/d | Average push force/N | |
| Example 1 | 217 | 10.4 |
| Example 2 | 267 | 13.7 |
| Example 3 | 264 | 13.5 |
| Example 4 | 268 | 13.8 |
| Comparative example 1 | 244 | 13.2 |
| Comparative example 2 | 235 | 12.1 |
| Comparative example 3 | 252 | 11.4 |
It is concluded from the comparison of the data in the table that the average pushing force of examples 2-4 and comparative examples 1-3 is slightly increased compared with example 1, but the pushing force is between 8 and 16N, and is still suitable for injection. Comparative example 1 does not carry out sulfhydrylation modification on sheep collagen fibers, and has the advantages of reduced crosslinking property and compatibility and reduced degradation time. Comparative example 2 was free of addition of thiolated hyaluronic acid and the degradation resistance was lowered. Comparative example 3 the concentration of hydrogen peroxide was 3 mmol/L, at which time the concentration of hydrogen peroxide became low, the stability of the material was lowered, the biodegradation rate was increased, and the degradation time was shortened. According to the embodiment 2-4, the sheep collagen fibers and the sulfhydryl hyaluronic acid are added, so that the degradation time can be prolonged, and a bracket space is provided for tissue regeneration. The sheep collagen fiber is subjected to sulfhydrylation modification, so that the sheep collagen fiber contains sulfhydryl groups, sulfhydryl groups exist on sulfhydrylation hyaluronic acid and silk fibroin freeze-dried particles, the three have good compatibility, and the degradation time is prolonged better. The concentration of hydrogen peroxide is controlled to be 5-5.5mmol/L, so that the stability of the material is improved, the biodegradation rate is reduced, and the degradation time is further prolonged.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (9)
1. A preparation method of a polymer filler containing silk fibroin particles is characterized by comprising the following steps:
Dissolving degummed silk with lithium bromide solution to obtain dissolved silk, cooling the dissolved silk, dialyzing, and taking out to obtain silk solution;
step two, taking a silk fibroin solution, filtering, centrifuging, concentrating at 60-65 ℃, and adding water for injection for dilution to obtain a diluted silk fibroin solution;
Thirdly, placing the diluted silk fibroin solution at 60-65 ℃ until silk fibroin gel is formed;
Step four, taking silk fibroin gel, granulating, adding concentrated silk fibroin solution into the granulated silk fibroin gel particles, uniformly mixing, freezing at-40 ℃ to-80 ℃ for 24-48 hours, freeze-drying, absorbing moisture at 25-30 ℃ for 1-3d, and crushing to obtain silk fibroin freeze-dried particles;
Mixing silk fibroin freeze-dried particles, sulfhydryl hyaluronic acid and deionized water uniformly, regulating the pH value of the system to 8-8.5 by using a sodium hydroxide solution, adding sulfhydryl sheep collagen fiber particles, mixing uniformly at 0-2 ℃, adding hydrogen peroxide, reacting for 20-30min at 35-40 ℃, dialyzing, freeze-drying to obtain particles A, taking the particles A and sodium hyaluronate gel, and mixing uniformly to obtain the polymer filler containing silk fibroin particles.
2. The method of claim 1, wherein the degummed silk and lithium bromide are used in a bath ratio of 1 (4-10).
3. The method of claim 1, wherein the concentration of the diluted silk fibroin solution in the second step is 2wt% to 6wt%.
4. The method of producing a polymer filler containing silk fibroin particles according to claim 1, wherein in the fourth step, the mass ratio of silk fibroin gel particles after granulation to silk fibroin in the concentrated silk fibroin solution is 1 (0.2-2).
5. The method of claim 1, wherein the step II is performed under a centrifugation condition of 9000rpm for 20 to 30 minutes.
6. The preparation method of the polymer filler containing the silk fibroin particles is characterized by comprising the steps of uniformly mixing sheep collagen fibers and acetic acid to obtain a sheep collagen fiber solution, cooling to 2-3 ℃, adding N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, uniformly mixing, reacting for 30-40min, then dropwise adding a cysteamine hydrochloride solution, adding a 2- (N-morpholino) ethane sulfonic acid solution, regulating the pH value of the solution to 5.5-6, stirring for 4-5h, reacting for 10-14h at 4-5 ℃, dialyzing to obtain the thiol-modified sheep collagen fiber solution, and freeze-drying to obtain the thiol-modified sheep collagen fiber particles.
7. The method for preparing the polymer filler containing the silk fibroin particles, as set forth in claim 6, characterized in that the method for preparing the sheep collagen fibers comprises the steps of dissolving sheep collagen powder in acetic acid, dialyzing to neutrality to obtain a sheep collagen solution, adding a phosphate buffer, uniformly mixing, centrifuging, then re-suspending the precipitate in the phosphate buffer, centrifuging, and drying to obtain the sheep collagen fibers.
8. The method for preparing the polymer filler containing the silk fibroin particles, as set forth in claim 7, characterized in that the method for preparing the sheep collagen powder comprises the steps of taking sheep skin, breaking, washing, degreasing by using n-butanol, centrifuging, washing, extracting collagen by using acetic acid solution containing pepsin, adjusting the pH value to be neutral, inactivating enzyme, dialyzing, and freeze-drying to obtain the sheep collagen powder.
9. The method of claim 1, wherein the concentration of hydrogen peroxide is 5-5.5mmol/L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202510053643.0A CN119770747B (en) | 2025-01-14 | 2025-01-14 | A method for preparing a polymer filler containing silk fibroin particles |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202510053643.0A CN119770747B (en) | 2025-01-14 | 2025-01-14 | A method for preparing a polymer filler containing silk fibroin particles |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN119770747A CN119770747A (en) | 2025-04-08 |
| CN119770747B true CN119770747B (en) | 2025-07-11 |
Family
ID=95244150
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202510053643.0A Active CN119770747B (en) | 2025-01-14 | 2025-01-14 | A method for preparing a polymer filler containing silk fibroin particles |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN119770747B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111440340A (en) * | 2020-04-30 | 2020-07-24 | 苏州苏豪生物材料科技有限公司 | A kind of silk fibroin-sodium hyaluronate cross-linked double network gel and preparation method thereof |
| CN116217943A (en) * | 2023-03-03 | 2023-06-06 | 浙江省人民医院 | A photocrosslinked silk fibroin/hyaluronic acid hydrogel barrier material and its preparation method and application |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110008436A1 (en) * | 2009-04-20 | 2011-01-13 | Altman Gregory H | Silk Fibroin Hydrogels and Uses Thereof |
| KR101629204B1 (en) * | 2014-08-19 | 2016-06-10 | 전북대학교산학협력단 | Thin Film Silk fibroin/collagen composite implant and Preparation Method Thereof |
-
2025
- 2025-01-14 CN CN202510053643.0A patent/CN119770747B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111440340A (en) * | 2020-04-30 | 2020-07-24 | 苏州苏豪生物材料科技有限公司 | A kind of silk fibroin-sodium hyaluronate cross-linked double network gel and preparation method thereof |
| CN116217943A (en) * | 2023-03-03 | 2023-06-06 | 浙江省人民医院 | A photocrosslinked silk fibroin/hyaluronic acid hydrogel barrier material and its preparation method and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN119770747A (en) | 2025-04-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Liang et al. | Injectable protocatechuic acid based composite hydrogel with hemostatic and antioxidant properties for skin regeneration | |
| Yan et al. | Facile preparation of bioactive silk fibroin/hyaluronic acid hydrogels | |
| EP2892575B1 (en) | Hyaluronic acid/collagen- based dermal filler compositions and methods for making same | |
| US20230201418A1 (en) | Soft tissue augmentation using injectable, neutral ph soluble collagen-glycosaminoglycan compositions | |
| CN111440340B (en) | A kind of silk fibroin-sodium hyaluronate cross-linked double network gel and preparation method thereof | |
| KR20200036664A (en) | Hyaluronic acid-silk fibroin hybrid hydrogels, and preparation method thereof | |
| CN112812329B (en) | Hydrogel of sulfhydryl modified high molecular compound, preparation method and application thereof | |
| CN119770740B (en) | A preparation method of silk fibroin-hyaluronic acid filler for injection | |
| Chen et al. | Functionalized gelatin/strontium hydrogel bearing endothelial progenitor cells for accelerating angiogenesis in wound tissue regeneration | |
| CN119215228B (en) | A controllable injectable silk fibroin extracellular matrix gel material and its preparation method | |
| CN113877001A (en) | Silk fibroin composite gel for injection | |
| CN118767212B (en) | Preparation of a porous artificial bone composite of silk fibroin hydroxyapatite and hyaluronic acid | |
| Cai et al. | Enzyme-crosslinked hyaluronic acid hydrogel scaffolds for BMSCs microenvironment and wound healing | |
| WO2016100476A1 (en) | Dermal fillers comprising keratin biomaterials | |
| CN119770747B (en) | A method for preparing a polymer filler containing silk fibroin particles | |
| Qian et al. | Regulation of physicochemical properties of alginate-based hydrogels and preliminary applications in wound healing | |
| TWI880094B (en) | Cross-linking polysaccharide with fibroin and uses of the obtained material | |
| CN115192776B (en) | Method for preparing tough hydrogel for repairing tendon injury | |
| KR102385695B1 (en) | Novel hyaluronic acid -amino hydrogel complex composition with semi-IPN structure for soft tissue repair and augmentation and an use thereof | |
| KR102417671B1 (en) | Hyaluronic acid-based filler composition having self-crosslinking system and method for manufacturing the same | |
| CN120617619A (en) | Silk fibroin gel and preparation method thereof | |
| EP3765103A1 (en) | Porous biomaterials for tissue regeneration | |
| CN109010912B (en) | Modified hyaluronic acid injectable filling material and preparation method thereof | |
| CN119868661B (en) | A hyaluronic acid gel mixture and its preparation method and application | |
| CN119405890B (en) | Cartilage repair hydrogel material and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |