CN113209378A - Injectable silk fibroin/nano hydroxyapatite composite hydrogel and preparation method thereof - Google Patents

Injectable silk fibroin/nano hydroxyapatite composite hydrogel and preparation method thereof Download PDF

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CN113209378A
CN113209378A CN202110573591.1A CN202110573591A CN113209378A CN 113209378 A CN113209378 A CN 113209378A CN 202110573591 A CN202110573591 A CN 202110573591A CN 113209378 A CN113209378 A CN 113209378A
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silk fibroin
hydroxyapatite
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肖芸
龙仕和
吴永豪
朱向东
张兴栋
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Sichuan University
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces

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Abstract

The invention provides injectable silk fibroin/nano hydroxyapatite composite hydrogel and a preparation method thereof, wherein the preparation method comprises the following steps: firstly degumming and drying silkworm cocoons to obtain silk fibroin, dissolving the silk fibroin by a solvent at a certain temperature, mixing the silk fibroin and phosphate to obtain a mixed solution, adjusting the pH value of the mixed solution, reacting for a period of time, dialyzing, drying to obtain silk fibroin/nano-hydroxyapatite powder, and then carrying out ultrasonic treatment to generate the silk fibroin/nano-hydroxyapatite composite hydrogel. The mass volume concentration of the silk fibroin/hydroxyapatite powder is 0.1-90%; the silk fibroin: the mass ratio of the hydroxyapatite is 5-99: 1-95. The composite hydrogel prepared by the invention has the advantages of uniformity, good interface combination, no bubble in the interior, safety, no toxicity and excellent injectability; the preparation method has the advantages of simple preparation process, mild conditions, no chemical cross-linking agent in the whole process, environmental protection, low cost and easy industrialization, popularization and application.

Description

Injectable silk fibroin/nano hydroxyapatite composite hydrogel and preparation method thereof
Technical Field
The invention relates to the technical field of biomedical materials, in particular to injectable silk fibroin/nano hydroxyapatite composite hydrogel and a preparation method thereof.
Background
The hydrogel is a cross-linked polymer with a three-dimensional network structure, can well simulate the water environment of extracellular matrix, provides a microenvironment similar to natural extracellular matrix for the adhesion, proliferation and differentiation of cells, is an ideal material for tissue injury regeneration, repair and reconstruction, is widely applied to the biomedical field, such as medical dressings, cartilage tissue repair, cell transplantation, drug delivery, tissue engineering scaffolds and the like, and particularly has the greatest attraction for the injectable hydrogel for tissue repair, can be transplanted to a defect part in a minimally invasive mode, can also fill defects in any shape, and is convenient for carrying cells and bioactive factors, thereby attracting great attention of people.
Hydroxyapatite (HA, chemical formula Ca)10(OH)2(PO4)6) Is an important component of human bone tissue, can form chemical bonding with the human bone tissue, has excellent biocompatibility and osteoinductivity, is a typical bioactive material, and is widely applied to the fields of hard tissue repair, soft tissue repair, drug controlled release and the like. Common methods for preparing hydroxyapatite include hydrothermal synthesis, chemical precipitation, microemulsion, and sol-gel methods. However, with the rapid development of modern medicine and personalized treatment, the formed hydroxyapatite crystals have low strength, small porosity, poor osteoinduction and conductivity, and the defects of large brittleness, difficult processability and the like exist when the hydroxyapatite crystals are further prepared into a ceramic scaffold form for application, so that the hydroxyapatite crystals are far from meeting clinical requirements, and the application is greatly limited. Thus, HAP biocomposites have been prepared by various methods to improve their properties, and are usually prepared as composites with polymeric materialsThe hydrogel can overcome the defects of high brittleness, difficult molding and the like of hydroxyapatite, can improve the biocompatibility and bioactivity of a polymer material, has the advantages of promoting vascularization formation, remineralization and the like of the surface of a bone, and can ensure that new cells and nutrients can enter the inner side of the scaffold through the three-dimensional network pore structure of the hydrogel to induce osteogenesis so as to better finish the repair of bone defects.
Silk fibroin is an ideal natural polymer material and is often selected as a carrier material such as hydroxyapatite and the like. In recent years, there are many ways to prepare silk fibroin/hydroxyapatite composite materials. For example: (1) directly compounding hydroxyapatite powder and silk fibroin solution, and freeze-drying to form a scaffold or preparing composite hydrogel by adopting modes such as crosslinking and the like; (2) obtaining hydroxyapatite and silk fibroin nanofiber through electrostatic spinning; (3) the silk fibroin film is used for adsorbing hydroxyapatite to obtain the composite material. However, the method is essentially physical mixing and is not a combination of chemical bonds, and the interface combination strength, mixing scale and uniformity are difficult to guarantee. In addition, the regenerated silk fibroin solution is extremely unstable, and can spontaneously form gel along with the lapse of time even if being stored at low temperature, and the regenerated silk fibroin solution is best to be prepared and used, so that great inconvenience is brought to the preparation, transportation and storage of the silk fibroin/hydroxyapatite composite hydrogel, and the preparation process is very complicated, thereby limiting the clinical popularization and application and the efficiency exertion of the regenerated silk fibroin solution. Hydroxyapatite is often used as a material for injectable hydrogel due to its good biocompatibility and osteoinductivity, but the hydrogel products prepared from hydroxyapatite currently face many problems: the hydroxyapatite powder is easy to agglomerate due to small particle size, which causes the problems of uneven distribution of inorganic phases in the composite material, difficult removal of bubbles, difficult storage and transportation of hydrogel raw materials and the like.
Therefore, the innovative preparation method of the hydroxyapatite is used for developing novel hydroxyapatite hydrogel so as to meet the great requirements of the development of new materials and related industries in China, particularly the aspects of biological medicines, tissue engineering and the like, and the novel hydroxyapatite hydrogel becomes a hotspot for research in the field of tissue engineering and is a problem to be solved urgently.
Disclosure of Invention
The invention mainly solves the technical problem of providing injectable silk fibroin/nano hydroxyapatite composite hydrogel which has good inorganic phase uniformity, no bubbles and good interface combination and can meet the requirement of mechanical strength.
In order to solve the technical problems, the invention adopts the following technical scheme:
the invention provides silk fibroin/nano hydroxyapatite powder, which comprises silk fibroin and nano hydroxyapatite;
the mass volume concentration of the silk fibroin/hydroxyapatite powder is 0.1-90%; further, the silk fibroin: the mass ratio of the hydroxyapatite is 5-99: 1-95.
In a specific embodiment of the present invention, the hydroxyapatite is one of hydroxyapatite nanoparticles and hydroxyapatite nanorods, preferably hydroxyapatite nanoparticles; further, the particle size of the hydroxyapatite nano particle is 50-100 nm;
the silk fibroin is obtained by degumming silkworm cocoons;
further, the reagent for degumming is sodium salt solution, and the sodium salt solution is selected from Na2CO3、NaHCO3One of the solutions, preferably Na2CO3A solution; further 0.01 to 2% of Na2CO3Solution, preferably 0.05% Na2CO3And (3) solution.
The invention also provides a preparation method of the silk fibroin/hydroxyapatite powder, which comprises the following steps: dissolving silk fibroin by using a solvent, mixing the dissolved silk fibroin with phosphate to obtain a mixed solution, reacting, dialyzing to obtain a silk fibroin/nano-hydroxyapatite composite solution system, and drying to obtain the silk fibroin/nano-hydroxyapatite composite solution.
Methods of drying include, but are not limited to, vacuum freeze drying, vacuum drying, spray drying, and drying by methods known in the art may be suitable for use in the present invention.
In a specific embodiment of the invention, the mass ratio of the silk fibroin to the phosphate is 2: 0.5-6, preferably 1: 0.5-3.
In a particular embodiment of the invention, the solvent is a ternary calcium salt/alcohol/water solution, wherein the ratio of calcium salt: alcohol: the molar ratio of water is 1:2: 8;
the calcium salt is selected from one of calcium chloride and calcium nitrate, preferably calcium chloride; the alcohol is ethanol;
the dissolving temperature is 65-80 ℃;
the mass volume concentration of the dissolved silk fibroin solution is 0.1-50%.
In a particular embodiment of the invention, the phosphate is selected from NH4H2PO4、Na2HPO4、(NH4)2HPO4、Na3PO4、K2HPO4Is preferably (NH)4)2HPO4。
In a specific embodiment of the invention, the molar ratio of calcium to phosphorus in the mixed solution is 8-12: 4-7, preferably 10: 6;
the pH value of the mixed solution is 8-12;
the mixing temperature is 20-80 ℃.
The pH value of the mixed solution can be adjusted by sodium hydroxide, ammonia water and potassium hydroxide.
In a specific embodiment of the invention, the reaction time is 1-24 h.
Because the scaffold is formed by simply freeze-drying the silk fibroin and hydroxyapatite composite, the forming effect of the scaffold is poor, the scaffold has the defects of fragility, difficult forming and the like, the viscosity of the whole mixture system is increased by using carboxymethyl cellulose in the prior art so as to increase the forming effect of the scaffold, but even if the scaffold is manufactured, the defects of high brittleness, difficult processing performance and the like still exist in the application process, the application range is narrow, the scaffold is only limited in regular tissue defects, actually, injuries in human tissues such as arthritis, articular cartilage defects, fracture and the like are irregular, the scaffold material cannot meet the requirements, but the hydrogel material can meet the requirements of defect filling in any shape.
The invention also provides injectable silk fibroin/nano hydroxyapatite composite hydrogel which is obtained by dissolving the silk fibroin/hydroxyapatite powder and then carrying out ultrasonic treatment;
or the silk fibroin/nano hydroxyapatite composite solution system is subjected to ultrasonic treatment to obtain the silk fibroin/nano hydroxyapatite composite solution.
Further, the mass volume concentration of the dissolved silk fibroin/hydroxyapatite is 0.1-90%;
the ultrasonic treatment conditions are as follows: the ultrasonic power is 250 watts, the ultrasonic power ratio is 1-100%, and the time is 1 s-10 min.
The mass-to-volume concentration (w/v) of the present invention is the mass (g) concentration of a certain component in a unit volume (mL) of a mixture, i.e., the ratio of the mass (g) of a certain component in a mixture to the volume (mL) of the mixture.
The invention also provides an application method of the injectable silk fibroin/nano hydroxyapatite composite hydrogel, which comprises but is not limited to injecting the composite hydrogel into a defect part for in-situ gelling; or injecting the gel into the defect part after the gel is formed.
The invention has the beneficial effects that:
(1) the silk fibroin/nano hydroxyapatite composite hydrogel takes silk fibroin as a raw material, fully utilizes calcium ions in a ternary system as a calcium source to synthesize hydroxyapatite in situ, saves cost and achieves the aim of changing waste into valuable.
(2) The silk fibroin/nano hydroxyapatite composite solution system obtained by dialysis after reaction has gel characteristics, can meet the filling requirement of any defect shape, can obtain injectable composite hydrogel by selecting a green and environment-friendly ultrasonic method to induce the silk fibroin/nano hydroxyapatite composite solution system, has high efficiency and rapidness in preparation method, avoids using a chemical cross-linking agent in the whole process, is simple to operate and mild in condition, does not need complex equipment and facilities, and is easy to industrialize, popularize and apply.
(3) According to the invention, calcium ions and silk fibroin are combined through electrostatic adsorption, and the combination is a chemical force combination, so that the obtained silk fibroin/nano hydroxyapatite composite hydrogel has good inorganic phase uniformity and interface binding force, has no foaming phenomenon in the interior, and can better meet the clinical application requirements.
(4) The silk fibroin/nano hydroxyapatite composite solution system can be continuously dissolved in water after being dried and still has the gel property, and the SF-nHA injectable hydrogel can be obtained again by ultrasonic treatment and other methods, so that the bottleneck problems of raw material transportation, storage and the like are solved.
(5) Under the condition that ultrasonic treatment parameters, silk fibroin and nano hydroxyapatite are the same in proportion, an 18G needle is used for detecting that the injectability of the SF-nHA hydrogel (in-situ synthesis and injection thrust of less than 25N) prepared by the method is far better than that of the SF-nHA hydrogel (physical mixing and injection thrust of more than 80N); the SF-nHA hydrogel still shows good injectability by using a 27G needle, and the injection thrust is not more than 45N; the SF-nHA hydrogel can also improve the injectability by adjusting ultrasonic parameters, the percentage concentration of the SF-nHA, the proportion of the SF-nHA and the like according to the actual application requirements.
Drawings
FIG. 1 shows the dry powder state of SF-nHA obtained in example 1;
FIG. 2 is a scanning electron micrograph of the SF-nHA powder obtained in example 1;
FIG. 3 is a scanning electron microscope image of freeze-drying after physical mixing of nHA powder and silk fibroin solution;
FIG. 4 is the XRD patterns of dry SF-nHA powder and HA (09-0432) obtained in example 1;
FIG. 5 shows the molded state of the appearance of the composite hydrogel obtained in example 1;
FIG. 6 is a comparison of the injectability of the SF-nHA hydrogel (synthesized in situ) and SF-nHA hydrogel (physically mixed) of example 1 after gelling using an 18G needle.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
Example 1
(1) 0.05% of Na is selected2CO3Degumming 20g of silkworm cocoons by using 1L of solution, washing and drying to obtain the silk fibroin fibers.
(2) At 70 ℃, 50ml of calcium chloride/ethanol/water (molar ratio is 1:2: 8) ternary system solution is used for dissolving 5g of silk fibroin fibers, and 10% (w/v) silk fibroin mixed solution is prepared.
(3) Gradually adding 10.0g of ammonium dihydrogen phosphate into the silk fibroin mixed solution obtained in the step (2) according to the molar ratio of calcium to phosphorus of 10:6 at 37 ℃, adjusting the pH value to 10.0 by using 2M ammonia water, and stirring for reacting for 6 h; after the reaction is finished, the silk fibroin/nano hydroxyapatite composite solution is obtained through dialysis.
(4) Performing ultrasonic induction on the silk fibroin/nano hydroxyapatite composite solution obtained in the step (3) for 1min by using ultrasonic power of 250 watts and ultrasonic power ratio of 90 percent to obtain injectable silk fibroin/nano hydroxyapatite composite hydrogel; or performing vacuum freeze drying on the silk fibroin/nano hydroxyapatite composite solution obtained in the step (3) to obtain SF-nHA (silk fibroin-nano hydroxyapatite) powder with good dispersibility, wherein the mass percent of SF: HA 4.5: 9.3, fully dissolving 4.0gSF-nHA powder in 15ml of pure water to prepare 26.7 percent (w/v) solution, and carrying out ultrasonic induction for 1min by using ultrasonic power of 250 watts and ultrasonic power ratio of 90 percent to obtain the injectable silk fibroin/nano hydroxyapatite composite hydrogel.
Example 2
(1) 0.05% of Na is selected2CO3Degumming 20g of silkworm cocoons by using 1L of solution, washing and drying to obtain the silk fibroin fibers.
(2) At 70 ℃, dissolving 15g of silk fibroin fiber by using 50ml of calcium chloride/ethanol/water (molar ratio is 1:2: 8) ternary system solution to prepare 15% (w/v) silk fibroin mixed solution.
(3) Gradually adding 15.0g of diammonium phosphate into the silk fibroin mixed solution obtained in the step (2) according to the molar ratio of calcium to phosphorus of 10:6 at 60 ℃, adjusting the pH value to 9.0 by using 2M sodium hydroxide, and stirring for reacting for 4 hours; after the reaction is finished, the silk fibroin/nano hydroxyapatite composite solution is obtained through dialysis.
(4) Performing ultrasonic induction on the silk fibroin/nano-hydroxyapatite composite solution obtained in the step (3) for 2min by using ultrasonic power of 250 watts and ultrasonic power ratio of 30 percent to obtain injectable silk fibroin/nano-hydroxyapatite composite hydrogel; or carrying out vacuum freeze drying on the silk fibroin/nano hydroxyapatite composite solution obtained in the step (3) to obtain SF-nHA powder with good dispersibility, wherein the mass percentage of SF: HA 13.5: 18.4, fully dissolving 2gSF-nHA powder in 5ml of pure water to prepare a 40% (w/v) solution, and performing ultrasonic induction for 2min by using ultrasonic power of 250 watts and ultrasonic power ratio of 30% to obtain the injectable silk fibroin/nano hydroxyapatite composite hydrogel.
Example 3
(1) 0.05% of Na is selected2CO3Degumming 20g of silkworm cocoons by using the solution, and washing and drying to obtain the silk fibroin fibers.
(2) At 75 ℃, dissolving 5g of silk fibroin fiber by using 100ml of a calcium chloride/ethanol/water ternary system solution (the molar ratio is 1:2: 8) to prepare 5% (w/v) silk fibroin mixed solution.
(3) Gradually adding 30g of diammonium phosphate into the silk fibroin mixed solution obtained in the step (2) according to the molar ratio of calcium to phosphorus of 10:6 at the temperature of 60 ℃, adjusting the pH value to 10 by using ammonia water, and stirring for reacting for 2 hours; after the reaction is finished, the silk fibroin/nano hydroxyapatite composite solution is obtained through dialysis.
(4) Performing ultrasonic induction on the silk fibroin/nano hydroxyapatite composite solution obtained in the step (3) for 0.5min by using ultrasonic power of 250 watts and an ultrasonic power ratio of 70 percent to obtain injectable silk fibroin/nano hydroxyapatite composite hydrogel; or carrying out vacuum freeze drying on the silk fibroin/nano hydroxyapatite composite solution obtained in the step (3) to obtain SF-nHA powder with good dispersibility, wherein the mass percentage of SF: HA 4.3: 36.9 and fully dissolving the 4gSF-nHA powder in 5ml of pure water to prepare a 20% (w/v) solution, and carrying out ultrasonic induction for 0.5min by using the ultrasonic power of 250 watts and the ultrasonic power ratio of 70 percent to obtain the injectable silk fibroin/nano hydroxyapatite composite hydrogel.
As can be seen from FIG. 1, the SF-nHA powder obtained by the method of example 1 was white and had good dispersibility;
FIG. 2 shows that the nano-hydroxyapatite is uniformly distributed on the silk fibroin material by a scanning electron microscope, and the interface bonding is good;
as can be seen from fig. 3, SF-nHA obtained by physical mixing is not uniform, and the nano hydroxyapatite and the silk fibroin material have separation signs;
as can be seen from FIG. 4, the silk fibroin/nano hydroxyapatite composite solution after ultrasonic treatment was injected into the patient
Figure BDA0003083455500000071
The silica gel mold is incubated for 30min at 37 ℃, and the gelling and forming effects are better; from the XRD pattern of figure 4, the SF-nHA powder keeps highly consistent with the characteristic peak of hydroxyapatite standard card (HA09-0432), which shows that the nano hydroxyapatite synthesized by the method HAs high crystallinity and purity;
as can be seen from FIG. 5, the silk fibroin/nano hydroxyapatite composite solution after ultrasonic treatment was injected into the patient
Figure BDA0003083455500000072
The silica gel mold is incubated for 30min at 37 ℃, and the gelling and forming effects are better;
fig. 6 is an injectable hydrogel obtained by further dissolving the SF-nHA (silk fibroin-nano hydroxyapatite) powder obtained by vacuum freeze drying in the step (3) in water, performing ultrasonic treatment, transferring the solution to an injector, and incubating the solution for 30min, wherein the injectable properties of the SF-nHA hydrogel (in situ synthesis) and the SF-nHA hydrogel (physical mixing) are tested by using an 18G needle under the condition that the ultrasonic treatment parameters and the proportion of the silk fibroin and the nano hydroxyapatite are the same, and the result shows that the injectable property effect of the SF-nHA hydrogel (in situ synthesis, the injection thrust is less than 25N) obtained by dissolving the solution after drying is far better than that of the SF-nHA hydrogel (physical mixing, the injection thrust is more than 80N). Moreover, the SF-nHA hydrogel (in-situ synthesized) still shows good injectability by using a 27G needle, and the injection thrust is not more than 45N.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims (10)

1. A silk fibroin/nano hydroxyapatite powder is characterized by comprising silk fibroin and nano hydroxyapatite;
the mass volume concentration of the silk fibroin/hydroxyapatite powder is 0.1-90%; further, the silk fibroin: the mass ratio of the hydroxyapatite is 5-99: 1-95.
2. The silk fibroin/hydroxyapatite powder according to claim 1, wherein the hydroxyapatite is one of hydroxyapatite nanoparticles and hydroxyapatite nanorods, preferably hydroxyapatite nanoparticles; further, the particle size of the hydroxyapatite nano particle is 50-100 nm;
the silk fibroin is obtained by degumming silkworm cocoons;
further, the reagent for degumming is sodium salt solution, and the sodium salt solution is selected from Na2CO3、NaHCO3One of the solutions, preferably Na2CO3A solution; further 0.01 to 2% of Na2CO3Solution, preferably 0.05% Na2CO3And (3) solution.
3. The method for preparing silk fibroin/hydroxyapatite powder according to claim 1 or 2, characterized by comprising: dissolving silk fibroin by using a solvent, mixing the dissolved silk fibroin with phosphate to obtain a mixed solution, reacting, dialyzing to obtain a silk fibroin/nano-hydroxyapatite composite solution system, and drying to obtain the silk fibroin/nano-hydroxyapatite composite solution.
4. The preparation method of claim 3, wherein the mass ratio of the silk fibroin to the phosphate is 1: 0.5-6, preferably 1: 0.5-3.
5. The method according to claim 3, wherein the solvent is a ternary calcium salt/alcohol/water solution, wherein the ratio of calcium salt: alcohol: the molar ratio of water is 1:2: 8;
the calcium salt is selected from one of calcium chloride and calcium nitrate, preferably calcium chloride; the alcohol is ethanol;
the dissolving temperature is 65-80 ℃;
the mass volume concentration of the dissolved silk fibroin solution is 0.1-50%.
6. The method according to claim 3, wherein the phosphate is selected from NH4H2PO4、Na2HPO4、(NH4)2HPO4、Na3PO4、K2HPO4Is preferably (NH)4)2HPO4。
7. The preparation method according to claim 3, wherein the calcium/phosphorus molar ratio in the mixed solution is 8-12: 4-7, preferably 10: 6;
the pH value of the mixed solution is 8-12;
the mixing temperature is 20-80 ℃.
8. The preparation method according to claim 3, wherein the reaction time is 1-24 hours.
9. An injectable silk fibroin/nano hydroxyapatite composite hydrogel is characterized in that the injectable silk fibroin/nano hydroxyapatite composite hydrogel is obtained by dissolving the silk fibroin/hydroxyapatite powder of claim 1 or 2 and then performing ultrasonic treatment;
or is obtained by carrying out ultrasonic treatment on the silk fibroin/nano hydroxyapatite composite solution system of claim 3.
10. The composite hydrogel according to claim 9, wherein the mass volume concentration of the dissolved silk fibroin/hydroxyapatite is 0.1-90%;
the ultrasonic treatment conditions are as follows: the ultrasonic power is 200-400 watts, the ultrasonic power ratio is 1-100%, and the time is 1 s-10 min.
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Citations (4)

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CN103432629A (en) * 2013-07-22 2013-12-11 苏州三和开泰花线织造有限公司 Preparation method for silk fibroin/hydroxyapatite composite material
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