CN110698866A - Ultrasonic-mediated silk fibroin composite collagen hydrogel and preparation method thereof - Google Patents
Ultrasonic-mediated silk fibroin composite collagen hydrogel and preparation method thereof Download PDFInfo
- Publication number
- CN110698866A CN110698866A CN201910985899.XA CN201910985899A CN110698866A CN 110698866 A CN110698866 A CN 110698866A CN 201910985899 A CN201910985899 A CN 201910985899A CN 110698866 A CN110698866 A CN 110698866A
- Authority
- CN
- China
- Prior art keywords
- collagen
- solution
- silk fibroin
- hydrogel
- silk
- 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.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
- C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
- C08J3/075—Macromolecular gels
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
- A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
- A61L27/48—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with macromolecular fillers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/52—Hydrogels or hydrocolloids
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2389/00—Characterised by the use of proteins; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2489/00—Characterised by the use of proteins; Derivatives thereof
Abstract
The invention discloses an ultrasonic-mediated silk fibroin composite collagen raw hydrogel and a preparation method thereof. The mass ratio of the silk fibroin to the collagen is 99: 1-1: 99. The ultrasonic-mediated fibroin composite collagen hydrogel prepared by the invention is safe, nontoxic, good in biocompatibility, short in gelation time, controllable in degradation time, excellent in mechanical property, simple in preparation process, energy-saving, environment-friendly, low in cost and easy to industrialize, popularize and apply, and is physically blended in the whole process.
Description
Technical Field
The invention relates to the field of hydrogel, in particular to ultrasonic-mediated silk fibroin composite collagen hydrogel and a preparation method thereof.
Background
Hydrogels are gels with a three-dimensional network structure formed by polymer chains that retain water in the interstitial spaces between macromolecules. The material 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, and is widely applied to the biomedical field, such as medical dressings, cartilage tissue repair, cell transplantation, drug delivery, tissue engineering scaffolds and the like. However, the current hydrogel products for tissue repair still face a plurality of problems, wherein the problems of insufficient mechanical properties, mismatched in vivo degradation rate and regeneration rate of new tissues and the like are particularly prominent.
The collagen hydrogel belongs to a natural protein material, not only has good biocompatibility and biodegradability, but also has excellent bioactivity, and has the same or homologous components with extracellular matrix components, so the collagen hydrogel also has a plurality of sites capable of being recognized and adhered by cells, and can provide a surface which is closer to a natural state for the cells. The collagen fiber has strong self-assembly capability, is easy to form gel under the conditions of proper temperature and pH, and can provide excellent microenvironment for cell adhesion, proliferation and differentiation. However, the problems of unstable components and low purity of collagen during extraction are easily caused, and the problems of insufficient mechanical properties, short in-vivo degradation time and the like generally exist in a simple collagen hydrogel, and particularly, the collagen hydrogel is easy to produce severe volume shrinkage in the culture process after loading cells, so that the nutrition supply and biological behavior of the cells are influenced, and the requirement of rapid development of tissue engineering is difficult to meet. A great deal of researchers are dedicated to improving the mechanical strength of the collagen hydrogel through chemical crosslinking modification and obtaining a certain effect, but the introduction and the residue of the crosslinking agent tend to reduce the biocompatibility of the material and cause certain toxic and side effects to cells, and the collagen hydrogel cannot have the characteristics of excellent biocompatibility, proper mechanical strength and degradability, specific cell and molecule responsiveness and the like. Therefore, the direct combination of other polymer materials and physical crosslinking will be an important way to solve the above-mentioned bottleneck problem.
Silk fibroin is cheap and easy to obtain, is safe and nontoxic, and is plain in the reputations of fibre queen. Research shows that the hydrogel has the same in vivo and in vitro biocompatibility as collagen, and has the characteristics of high mechanical strength, controllable biodegradation and conversion to stable beta-sheet crystal conformation spontaneously or under the promotion of external conditions (such as ultrasonic waves, pH or a cross-linking agent) to form the physical cross-linked hydrogel. The characteristics can well make up the defects of collagen, and provide a new idea for designing and preparing the composite hydrogel material. At present, the application research of silk fibroin at home and abroad is rapidly expanded from the traditional textile field to the fields of cell engineering, tissue engineering, drug slow release, precise medical treatment, implantable medical devices, normal temperature stable vaccines, 3D bioprinting and the like, and a series of research results are obtained. Of course, as a tissue engineering scaffold, pure silk fibroin hydrogel has good cell affinity, but also has the defects of poor flexibility, brittleness, narrow adjustable range of structure and performance and the like, and the application range of the silk fibroin hydrogel is greatly limited.
At present, the ultrasound-mediated silk fibroin composite collagen hydrogel is reported, and the phenomenon of phase separation which is easy to occur after collagen and silk fibroin solution are directly mixed is still an innovative elbow which troubles scientific researchers. Therefore, how to timely and efficiently integrate resources and exert the advantages of two natural protein materials and create a novel multifunctional composite hydrogel which has high added value and can effectively make up the defects of simple collagen or silk fibroin hydrogel so as to meet the huge requirements of the development of new materials and related industries in China, particularly the development of biological medicines, tissue engineering and the like, becomes a hotspot of research in the field of tissue engineering and is urgently needed to be solved.
Disclosure of Invention
The invention mainly solves the technical problem of providing the ultrasonic-mediated silk fibroin composite collagen water gel which has excellent biocompatibility and mechanical property and small volume shrinkage change in the cell culture process.
In order to solve the technical problems, the invention adopts a technical scheme that:
providing an ultrasonic-mediated fibroin composite collagen water gel, which comprises fibroin protein and collagen protein; the ultrasonic-mediated silk fibroin composite collagen hydrogel is characterized in that the mass volume concentration of silk fibroin is 0.01-50%, and the mass volume concentration of collagen is 0.01-50%; further, the silk fibroin: the mass ratio of the collagen is 99: 1-1: 99.
Further, the collagen is type I collagen, preferably bovine type I collagen.
In the invention, the silk fibroin is obtained by degumming one or more of mulberry silk, tussah silk, camphor silk and willow silk, but is not limited to the silk varieties, and other silk products capable of preparing the silk fibroin can be suitable for the invention.
The invention also provides a preparation method of the ultrasonic-mediated fibroin composite collagen hydrogel, which comprises the following steps: and carrying out ultrasonic treatment on the silk fibroin solution, mixing the silk fibroin solution with the collagen solution to obtain a mixed solution, and incubating the mixed solution to gelatinize the mixed solution to obtain the collagen.
Further, the mass volume concentration of the collagen solution is 0.01-50%.
The mass-to-volume concentration (w/v) is the mass (g) concentration of a component in a unit volume (mL) of a mixture, i.e., the ratio of the mass (g) of a component in a mixture to the volume (mL) of the mixture.
Further, the pH value of the collagen solution is 3-10.
Further, the mass volume concentration of the silk fibroin solution is 0.01% -50%.
Further, the pH value of the silk fibroin solution is 3-10.
Further, the mass ratio of the silk fibroin solution to the collagen solution is 99: 1-1: 99.
Further, the ultrasonic treatment conditions are as follows: the ultrasonic power is 5-3000 watts, and the ultrasonic power ratio is as follows: 1-100%, for 1 second-2 hours, at 0-60 deg.C.
Further, the incubation conditions are: the temperature is 0-60 ℃ and the time is 0-2 hours.
In a specific embodiment of the invention, the silk fibroin solution is obtained by dissolving degummed silk in a solvent and dialyzing; further, the solvent is selected from CaCl2/C2H5OH/H2O ternary solution, LiBr aqueous solution, CaCl2One or more of aqueous solutions; further, the CaCl2/C2H5OH/H2CaCl in O ternary solution2:C2H5OH:H2The molar ratio of O is 1:2:8, the concentration of the LiBr aqueous solution is 9.3mol/L, and the CaCl is2The mass volume concentration of the aqueous solution is 10-100%; furthermore, the cut-off molecular weight of the dialysis is 1000-12000 Da.
Further, the collagen solution is obtained by dissolving collagen in acetic acid solution, hydrochloric acid solution or deionized water and adjusting the pH value. The pH value can be adjusted by using common pH value adjusting agents such as hydrochloric acid, acetic acid, sodium hydroxide solution and the like.
The collagen solution and the silk fibroin solution in the present invention are not limited to the above-mentioned methods, and all of the collagen solution and the silk fibroin solution prepared by methods known in the art can be applied to the present invention.
The invention has the beneficial effects that:
(1) the ultrasonic-mediated silk fibroin composite collagen water gel adopts type I collagen and silk fibroin as raw materials, both of which are natural renewable protein materials, and has wide sources, low price and easy obtainment.
(2) Compared with the traditional gel prepared by simply self-assembling and crosslinking collagen, the composite hydrogel has the advantages that the in-vivo and in-vitro enzymolysis resistance is obviously improved, the degradation speed is reduced, the filling time of the composite hydrogel during tissue repair is prolonged, and the effectiveness of the material is improved.
(3) Compared with the pure silk fibroin hydrogel, the addition of the collagen obviously increases the water absorption, flexibility and filling effect of the material.
(4) The ultrasonic-mediated silk fibroin composite collagen hydrogel disclosed by the invention does not have a phase separation phenomenon, the preparation method is efficient and rapid, physical mixing is adopted in the whole process, no chemical cross-linking agent is added, the operation is simple, the condition is mild, complex facilities and equipment are not needed, the energy is saved, the environment is protected, the cost is low, and the industrialization, the popularization and the application are easy.
(5) The ultrasonic-mediated silk fibroin composite collagen hydrogel has mechanical and structural properties similar to those of human extracellular matrix, has good water content and permeability, and can provide a superior microenvironment for adhesion, spreading and growth of cells. Meanwhile, the problem of negative influence on cells caused by severe hydrogel shrinkage in the process of culturing the cells by using single collagen hydrogel can be effectively solved, and the hydrogel material has wide application prospect in the field of tissue engineering regeneration and repair.
(6) The ultrasonic-mediated silk fibroin composite collagen hydrogel has a good interpenetrating network structure (IPN structure) and excellent mechanical properties, silk fibroin is firmly embedded on a collagen fiber structure, the collagen fiber structure is maintained, and the whole hydrogel is solid and stable in structure. The composite hydrogel has the advantages of pure collagen and silk fibroin hydrogel, can make up the defects of the pure collagen and silk fibroin hydrogel, can better support the adhesion, proliferation and growth of bone marrow mesenchymal stem cells, and can provide a novel three-dimensional culture model for the directional differentiation of material-induced stem cells.
Drawings
FIG. 1 is a flow chart of a process for preparing an ultrasonic-mediated silk fibroin composite gel raw hydrogel according to the present invention;
FIG. 2 is a scanning electron microscope image of the silk fibroin-collagen hydrogel ultrasonically mediated in example 1;
FIG. 3 is a confocal laser mapping of BMSCs on ultrasound-mediated growth of silk fibroin-collagen hybrid hydrogel in example 1;
FIG. 4 is a scanning electron microscope image of BMSCs in the ultrasound-mediated attachment and growth of silk fibroin-collagen hybrid hydrogel of example 1;
FIG. 5 is a comparison of mechanical properties of three hydrogel materials of BMSCs in COL, COL + SF (NS), and COL + SF (S) of example 1;
FIG. 6 is the ultrasound-mediated aqueous hydrogel of silk fibroin composite collagen prepared in example 1 comparing the initial and first day hydrogel volume changes of BMSCs cultured with aqueous hydrogel of silk fibroin composite collagen and collagen;
fig. 7 shows the volume changes of the ultrasound-mediated silk fibroin composite hydrogel prepared in example 1 compared with the hydrogel volume changes of the silk fibroin composite collagen hydrogel and the collagen hydrogel three days before BMSCs are cultured.
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 derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The collagen used in the embodiment of the invention is bovine type I collagen.
Example 1 preparation of ultrasound-mediated Silk fibroin-composite collagen hydrogel
(1) Completely dissolving 0.45g of collagen in 1M 100mL of acetic acid solution at the temperature of 2-8 ℃, and adjusting the pH value to 7.0 to obtain a uniform collagen solution for later use.
(2) Dissolving 10g degummed silk in CaCl2/C2H5OH/H2Dissolving in ternary O (n ═ 1:2:8) solution at 75 +/-2 ℃ for 4h, dialyzing in deionized water for 3 days by using a dialysis bag with molecular weight cutoff of 3500Da after complete dissolution to obtain silk fibroin solution with the concentration of 5% (w/v), and diluting with deionized water to the concentration of 3% (w/v) for later use;
(3) measuring 50mL of the 5% silk fibroin solution prepared in the step (2), carrying out ultrasonic treatment on the silk fibroin solution for 1min at normal temperature by using ultrasonic power of 1000 Watts and an ultrasonic power ratio of 40%, and then placing the solution at 2-8 ℃ for later use;
(4) and (3) sequentially weighing 20mL of collagen solution and 10mL of silk fibroin solution from (1) and (3), fully and uniformly mixing to form a mixed solution, adjusting the pH to 7.0, pouring the mixed solution into a cylindrical container, and incubating at 37 ℃ for 30min to gelatinize the mixed solution, thereby obtaining the ultrasound-mediated silk fibroin composite collagen hydrogel.
The ultrasound-mediated silk fibroin composite collagen hydrogel prepared in this example was observed by a scanning electron microscope, as shown in fig. 2. As can be seen from FIG. 2, the composite hydrogel has a good interpenetrating network structure (IPN structure), and the silk fibroin is firmly embedded on the collagen fiber structure and has similar structural performance to human extracellular matrix.
Example 2 preparation of ultrasound-mediated Silk fibroin-composite collagen hydrogel
(1) Completely dissolving 1g of collagen in 0.3M 200mL of acetic acid solution at the temperature of 2-8 ℃, and adjusting the pH value to 5.0 to obtain a uniform collagen solution for later use.
(2) Dissolving 10g of degummed silk in 9.3MLiBr solution, dissolving for 4h at 60 +/-5 ℃, dialyzing for 3 days in deionized water by using a dialysis bag with the molecular weight cutoff of 1000Da after complete dissolution to obtain a silk fibroin solution with the concentration of 3% (w/v), and adjusting the pH value to 5 for later use;
(3) measuring 30mL of the 3% silk fibroin solution prepared in the step (2), treating the silk fibroin solution for 10 seconds at normal temperature with the ultrasonic power of 500 watts and the ultrasonic power ratio of 60%, and then placing the solution at 2-8 ℃ for later use;
(4) and (3) sequentially weighing 30mL of collagen solution and 10mL of silk fibroin solution from (1) and (3), fully and uniformly mixing to form a mixed solution, adjusting the pH to 7.2, pouring the mixed solution into a cylindrical container, and incubating at 37 ℃ for 20min to gelatinize the mixed solution, thereby obtaining the ultrasound-mediated silk fibroin composite collagen hydrogel.
Example 3 preparation of ultrasound-mediated Silk fibroin-composite collagen hydrogel
(1) Completely dissolving 8g of collagen in 0.3M 200mL of acetic acid solution at the temperature of 2-8 ℃, and adjusting the pH value to 5.0 to obtain a uniform collagen solution for later use.
(2) Dissolving 15g of degummed silk in 40% CaCl2Dissolving in the solution at 98 + -2 deg.C for 4h, dialyzing with a dialysis bag with cut-off molecular weight of 12000Da in deionized water for 3 days to obtain silk fibroin solution with concentration of 8% (w/v), and adjusting pH to 5;
(3) measuring 50mL of the 8% silk fibroin solution prepared in the step (2), treating the silk fibroin solution for 30 seconds under the conditions of an ultrasonic power of 500 watts, an ultrasonic power ratio of 60% and an ice bath, and then placing at 2-8 ℃ for later use;
(4) and (3) sequentially measuring 10mL of collagen solution and 10mL of silk fibroin solution from (1) and (3), fully and uniformly mixing to form a mixed solution, adjusting the pH to 7.0, pouring the mixed solution into a cylindrical container, and incubating at 37 ℃ for 5min to gelatinize the mixed solution, thereby obtaining the ultrasound-mediated silk fibroin composite collagen hydrogel.
Example 4 preparation of ultrasound-mediated Silk fibroin-composite collagen hydrogel
(1) Completely dissolving 1g of collagen in 1M 200mL of hydrochloric acid solution at the temperature of 2-8 ℃, and adjusting the pH value to 7.0 to obtain a uniform collagen solution for later use.
(2) Dissolving 10g of degummed silk in 9.3MLiBr solution, dissolving for 4h at 60 +/-5 ℃, dialyzing for 3 days in deionized water by using a dialysis bag with molecular weight cutoff of 8000Da after complete dissolution to obtain silk fibroin solution with the concentration of 5% (w/v), and adjusting the pH value to 7.0 for later use;
(3) measuring 60mL of the 5% silk fibroin solution prepared in the step (2), treating the silk fibroin solution for 5 seconds at normal temperature with the ultrasonic power of 2000 watts and the ultrasonic power ratio of 30%, and then placing at 2-8 ℃ for later use;
(4) and (3) sequentially weighing 30mL of collagen solution and 10mL of silk fibroin solution from (1) and (3), fully and uniformly mixing to form a mixed solution, adjusting the pH to 7.0, pouring the mixed solution into a cylindrical container, and incubating at 37 ℃ for 30min to gelatinize the mixed solution, thereby obtaining the ultrasound-mediated silk fibroin composite collagen hydrogel.
Example 5 preparation of ultrasound-mediated Silk fibroin-composite collagen hydrogel
(1) Completely dissolving 10g of collagen in 2M 200mL of hydrochloric acid solution at the temperature of 2-8 ℃, and adjusting the pH value to 7.0 to obtain a uniform collagen solution for later use.
(2) Dissolving 30g of degummed silk in 9.3MLiBr solution, dissolving for 4h at 60 +/-5 ℃, dialyzing for 3 days in deionized water by using a dialysis bag with molecular weight cutoff of 3500Da after complete dissolution to obtain silk fibroin solution with the concentration of 5% (w/v), and adjusting the pH value to 7.0 for later use;
(3) measuring 60mL of the 5% silk fibroin solution prepared in the step (2), treating the silk fibroin solution for 15 seconds under ice bath conditions with the ultrasonic power of 500 watts and the ultrasonic power ratio of 80%, and then placing at 2-8 ℃ for later use;
(4) and (3) sequentially weighing 30mL of collagen solution and 20mL of silk fibroin solution from (1) and (3), fully and uniformly mixing to form a mixed solution, adjusting the pH to 7.2, pouring the mixed solution into a cylindrical container, and incubating at 37 ℃ for 15min to gelatinize the mixed solution, thereby obtaining the ultrasound-mediated silk fibroin composite collagen hydrogel.
Example 6 preparation of ultrasound-mediated Silk fibroin-composite collagen hydrogel
(1) Completely dissolving 10g of collagen in 0.5M 200mL of hydrochloric acid solution at the temperature of 2-8 ℃, and adjusting the pH value to 5.0 to obtain a uniform collagen solution for later use.
(2) Dissolving 10g of degummed silk in 9.3MLiBr solution, dissolving for 4h at 60 +/-5 ℃, dialyzing for 3 days in deionized water by using a dialysis bag with the molecular weight cutoff of 12000Da after complete dissolution to obtain silk fibroin solution with the concentration of 5% (w/v), and adjusting the pH value to 5.0 for later use;
(3) measuring 50mL of the 5% silk fibroin solution prepared in the step (2), treating the silk fibroin solution for 3 minutes at normal temperature with the ultrasonic power of 1000 watts and the ultrasonic power ratio of 10%, and then placing at 2-8 ℃ for later use;
(4) and (3) sequentially weighing 30mL of collagen solution and 30mL of silk fibroin solution from (1) and (3), fully and uniformly mixing to form a mixed solution, adjusting the pH to 7.0, pouring the mixed solution into a cylindrical container, and incubating at 37 ℃ for 5min to gelatinize the mixed solution, thereby obtaining the ultrasound-mediated silk fibroin composite collagen hydrogel.
Example 7 preparation of ultrasound-mediated Silk fibroin-composite collagen hydrogel
(1) Completely dissolving 0.03g of collagen in 1M 100mL of acetic acid solution at the temperature of 2-8 ℃, and adjusting the pH value to 10.0 to obtain a uniform collagen solution for later use.
(2) Dissolving 50g of degummed silk inCaCl2/C2H5OH/H2Dissolving in ternary O (n ═ 1:2:8) solution at 75 + -2 deg.C for 4h, dialyzing with 3500Da molecular weight cutoff dialysis bag in deionized water for 3 days, adjusting pH to 3.0 to obtain 50% (w/v) silk fibroin solution;
(3) measuring 10mL of the 50% silk fibroin solution prepared in the step (2), treating the silk fibroin solution for 1min at normal temperature with the ultrasonic power of 1000 watts and the ultrasonic power ratio of 40%, and then placing at 2-8 ℃ for later use;
(4) and (3) sequentially weighing 20mL of collagen solution and 1mL of silk fibroin solution from (1) and (3), fully and uniformly mixing to form a mixed solution, adjusting the pH to 7.0, pouring the mixed solution into a cylindrical container, and incubating at 37 ℃ for 30min to gelatinize the mixed solution, thereby obtaining the ultrasound-mediated silk fibroin composite collagen hydrogel.
Example 8 preparation of ultrasound-mediated Silk fibroin-composite collagen hydrogel
(1) Completely dissolving 50g of collagen in 1M 100mL of acetic acid solution at the temperature of 2-8 ℃, and adjusting the pH value to 3.0 to obtain a uniform collagen solution for later use.
(2) Dissolving 10g of degummed silk in 9.3M LiBr solution, dissolving for 4h at 60 +/-5 ℃, dialyzing for 3 days in deionized water by using a 3500 molecular weight cut-off dialysis bag after complete dissolution to obtain 5% (w/v) silk fibroin solution, diluting to 0.03% (w/v) concentration by using deionized water, and adjusting pH to 5.0 for later use;
(3) measuring 50mL of the 0.03% silk fibroin solution prepared in the step (2), treating the silk fibroin solution for 3 minutes under the conditions of ultrasonic power of 1000 watts, ultrasonic power ratio of 10% and ice bath, and then placing at 2-8 ℃ for later use;
(4) and (3) sequentially weighing 1mL of collagen solution and 20mL of silk fibroin solution from (1) and (3), fully and uniformly mixing to form a mixed solution, adjusting the pH to 7.0, pouring the mixed solution into a cylindrical container, and incubating at 37 ℃ for 5min to gelatinize the mixed solution, thereby obtaining the ultrasound-mediated silk fibroin composite collagen hydrogel.
Example 9 preparation of ultrasound-mediated Silk fibroin-composite collagen hydrogel
(1) Completely dissolving 46g of collagen in 1M 100mL of acetic acid solution at the temperature of 2-8 ℃, and adjusting the pH value to 7.0 to obtain a uniform collagen solution for later use.
(2) Dissolving 45g of degummed silk in CaCl2/C2H5OH/H2Dissolving in ternary O (n ═ 1:2:8) solution at 75 + -2 deg.C for 4h, dialyzing in deionized water for 3 days with 3500 MWCO dialysis bag, adjusting pH to 10.0 to obtain 45% (w/v) fibroin protein solution;
(3) measuring 100mL of the 50% silk fibroin solution prepared in the step (2), treating the silk fibroin solution for 1min at normal temperature with the ultrasonic power of 1000 watts and the ultrasonic power ratio of 40%, and then placing at 2-8 ℃ for later use;
(4) and (3) sequentially weighing 0.2mL of collagen solution and 20mL of silk fibroin solution from (1) and (3), fully and uniformly mixing to form a mixed solution, adjusting the pH to 7.0, pouring the mixed solution into a cylindrical container, and incubating at 37 ℃ for 30min to gelatinize the mixed solution, thereby obtaining the ultrasound-mediated silk fibroin composite collagen hydrogel.
Example 10
Three groups of hydrogel materials, COL + SF (NS), and COL + SF (S), were prepared for performance testing. The COL group is collagen hydrogel, which is hydrogel only adopting collagen; the COL + SF (NS) group is fibroin and collagen composite hydrogel, which is hydrogel compounded with fibroin directly without ultrasonic treatment; COL + SF (S) group is the ultrasonic-mediated silk fibroin composite collagen hydrogel.
COL group: adjusting pH to 7.0 with collagen solution with collagen concentration of 3mg/ml, pouring into cylindrical container, and incubating at 37 deg.C for 30min for gelation to obtain collagen hydrogel.
COL + SF (NS) group: the preparation process is as in example 1, with the difference that: the ultrasonic treatment step of the step (3) is not needed; the concentration of collagen is 3mg/ml, and the concentration of silk fibroin is 10 mg/ml; the mass ratio of the collagen to the silk fibroin is 3: and 5, obtaining the fibroin and collagen composite hydrogel.
COL + SF (S) group: the ultrasound-mediated silk fibroin composite collagen hydrogel of the present invention prepared in example 1.
Three groups of materials were tested for mechanical properties with reference to prior art methods:
three groups of hydrogel materials, COL + SF (NS), and COL + SF (S), were placed in a dynamic mechanical tester (DMA) test bed to test the storage modulus of the hydrogel at a compression frequency of 1Hz at room temperature with the parameters of 5mN pre-stress, 20 μm amplitude, and 105% dynamic static force.
The test results are shown in fig. 5, in the case that the collagen concentration is kept unchanged, the introduction of silk fibroin can increase the storage modulus of the hydrogel, and the group COL + sf (ns) > COL, particularly when the silk fibroin solution is subjected to ultrasonic treatment, the hydrogel compounded with the collagen solution (COL + sf (s)) has a higher storage modulus than the group COL + sf (ns), and is significantly different from the group COL, which indirectly indicates that the hydrogel mechanical strength of the group COL + sf(s) is better than that of the group COL and the group of COL + sf (ns), and indicates that the hydrogel material of the group COL + sf(s) shows a significant advantage in resisting cell-mediated material shrinkage (the results of fig. 6 and 7 also indicate that the point is shown).
BMSCs (rabbit source) are packaged and cultured on three groups of materials according to a method in the prior art, the density of the BMSCs packaged by the three groups of materials is 500 ten thousand per ml, and the phenomenon is observed.
As shown in FIGS. 3-4, BMSCs can be well adhered, proliferated and grown in the ultrasound-mediated silk fibroin composite collagen hydrogel, and keep high activity.
As is apparent from FIGS. 6 and 7, the hydrogel materials of the COL group and the COL + SF (NS) group both showed significant shrinkage with the increase of the time for culturing BMSCs, while the volume of the inventive ultrasound-mediated silk fibroin-compounded collagen hydrogel, i.e., the COL + SF (S) group, was less changed during the cell culture process, and the diameters of the COL, COL + SF (NS), and COL + SF (S) groups were sequentially reduced by 88.0%, 88.5%, and 12.3% by the 3 rd day.
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. An ultrasonic-mediated silk fibroin composite collagen hydrogel is characterized by comprising silk fibroin and collagen;
the mass volume concentration of the silk fibroin is 0.01-50%, and the mass volume concentration of the collagen is 0.01-50%; further, the silk fibroin: the mass ratio of the collagen is 99: 1-1: 99.
2. The ultrasound-mediated silk fibroin composite collagen hydrogel of claim 1, wherein said collagen is type I collagen, preferably bovine-derived type I collagen;
further, the silk fibroin is obtained by degumming one or more of mulberry silk, tussah silk, camphor silk and willow silk.
3. The preparation method of the ultrasound-mediated silk fibroin composite collagen hydrogel as claimed in claim 1 or 2, which comprises the following steps: and carrying out ultrasonic treatment on the silk fibroin solution, mixing the silk fibroin solution with the collagen solution to obtain a mixed solution, and incubating the mixed solution to gelatinize the mixed solution to obtain the collagen.
4. The preparation method according to claim 3, wherein the collagen solution has a mass volume concentration of 0.01 to 50%; further, the pH value of the collagen solution is 3-10.
5. The preparation method of claim 3, wherein the mass volume concentration of the silk fibroin solution is 0.01% -50%; further, the pH value of the silk fibroin solution is 3-10.
6. The preparation method of any one of claims 3 to 5, wherein the mass ratio of the silk fibroin solution to the collagen solution is 99: 1-1: 99.
7. The production method according to claim 3, wherein the ultrasonic treatment conditions are: the ultrasonic power is 5-3000 watts, and the ultrasonic power ratio is as follows: 1-100%, for 1 second-2 hours, at 0-60 deg.C.
8. The method of claim 3, wherein the incubation conditions are: the temperature is 0-60 ℃ and the time is 0-2 hours.
9. The method of claim 3, wherein the silk fibroin solution is obtained by dissolving degummed silk in a solvent and dialyzing; further, the solvent is selected from CaCl2/C2H5OH/H2O ternary solution, LiBr aqueous solution, CaCl2One or more of aqueous solutions; further, the CaCl2/C2H5OH/H2CaCl in O ternary solution2:C2H5OH:H2The molar ratio of O is 1:2:8, the concentration of the LiBr aqueous solution is 9.3mol/L, and the CaCl is2The mass volume concentration of the aqueous solution is 10-100%; furthermore, the cut-off molecular weight of the dialysis is 1000-12000 Da.
10. The method according to claim 3, wherein the collagen solution is prepared by dissolving collagen in acetic acid solution, hydrochloric acid solution or deionized water, and adjusting pH.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910985899.XA CN110698866A (en) | 2019-10-17 | 2019-10-17 | Ultrasonic-mediated silk fibroin composite collagen hydrogel and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910985899.XA CN110698866A (en) | 2019-10-17 | 2019-10-17 | Ultrasonic-mediated silk fibroin composite collagen hydrogel and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110698866A true CN110698866A (en) | 2020-01-17 |
Family
ID=69201107
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910985899.XA Pending CN110698866A (en) | 2019-10-17 | 2019-10-17 | Ultrasonic-mediated silk fibroin composite collagen hydrogel and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110698866A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112778773A (en) * | 2021-03-10 | 2021-05-11 | 嘉兴学院 | 4D printing ink and preparation method thereof |
| CN114621465A (en) * | 2022-05-17 | 2022-06-14 | 天新福（北京）医疗器材股份有限公司 | Collagen hydrogel and preparation method thereof |
| CN114634762A (en) * | 2022-03-17 | 2022-06-17 | 东莞市人民医院 | Metal ion mediated protein coating, preparation method and application thereof |
| CN114848893A (en) * | 2022-04-08 | 2022-08-05 | 大连医科大学 | Silk fibroin-combined decellularized small intestine submucosa extracellular matrix hydrogel for pigs, and preparation method and application thereof |
| CN115368744A (en) * | 2022-09-13 | 2022-11-22 | 大连理工大学 | Preparation and application of fibroin and structural protein polymer composite nanoparticles |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101502670A (en) * | 2009-03-05 | 2009-08-12 | 苏州大学 | Method for preparing silk fibroin hydrogel |
| CN102688525A (en) * | 2012-05-07 | 2012-09-26 | 东南大学 | Bio-macromolecular hydrogel and preparation method thereof |
| CN106084257A (en) * | 2016-06-06 | 2016-11-09 | 东华大学 | A kind of composite aquogel and preparation method thereof |
-
2019
- 2019-10-17 CN CN201910985899.XA patent/CN110698866A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101502670A (en) * | 2009-03-05 | 2009-08-12 | 苏州大学 | Method for preparing silk fibroin hydrogel |
| CN102688525A (en) * | 2012-05-07 | 2012-09-26 | 东南大学 | Bio-macromolecular hydrogel and preparation method thereof |
| CN106084257A (en) * | 2016-06-06 | 2016-11-09 | 东华大学 | A kind of composite aquogel and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| JENNIFER O.BUITRAGO 等: "Silk fibroin/collagen protein hybrid cell-encapsulating hydrogels with tunable gelation and improved physical and biological properties", 《ACTA BIOMATERIALIA》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112778773A (en) * | 2021-03-10 | 2021-05-11 | 嘉兴学院 | 4D printing ink and preparation method thereof |
| CN112778773B (en) * | 2021-03-10 | 2022-05-10 | 嘉兴学院 | 4D printing ink and preparation method thereof |
| CN114634762A (en) * | 2022-03-17 | 2022-06-17 | 东莞市人民医院 | Metal ion mediated protein coating, preparation method and application thereof |
| CN114848893A (en) * | 2022-04-08 | 2022-08-05 | 大连医科大学 | Silk fibroin-combined decellularized small intestine submucosa extracellular matrix hydrogel for pigs, and preparation method and application thereof |
| CN114621465A (en) * | 2022-05-17 | 2022-06-14 | 天新福（北京）医疗器材股份有限公司 | Collagen hydrogel and preparation method thereof |
| CN115368744A (en) * | 2022-09-13 | 2022-11-22 | 大连理工大学 | Preparation and application of fibroin and structural protein polymer composite nanoparticles |
| CN115368744B (en) * | 2022-09-13 | 2023-09-12 | 大连理工大学 | Preparation and application of fibroin and structural protein polymer composite nano-particles |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110698866A (en) | Ultrasonic-mediated silk fibroin composite collagen hydrogel and preparation method thereof | |
| CN111303459B (en) | Preparation method of hyaluronic acid based double-crosslinked hydrogel | |
| Yan et al. | Injectable alginate/hydroxyapatite gel scaffold combined with gelatin microspheres for drug delivery and bone tissue engineering | |
| US11511018B2 (en) | Conductive biomimetic skin scaffold material with self-repairing function and a method of preparing the same | |
| CN109734851B (en) | Temperature-sensitive polymer, synthesis method thereof and temperature-sensitive injectable hydrogel | |
| CN110964215B (en) | Preparation method of L-polylactic acid and cross-linked hyaluronic acid composite gel for injection and obtained product | |
| CN112062981B (en) | Preparation method of culture medium mediated crosslinked hyaluronic acid-based double-crosslinked hydrogel | |
| CN101824160A (en) | Preparation method of chitosan/polyvinyl alcohol/polylactic acid blended porous membrane | |
| Wang et al. | Dual-enzymatically crosslinked and injectable hyaluronic acid hydrogels for potential application in tissue engineering | |
| CN103083735A (en) | Composite membrane for guiding bone tissue regeneration and preparation method thereof | |
| CN102784414A (en) | Preparation method of injectable double-cross-linked hydrogel for tissue engineering | |
| CN111962210B (en) | Polycaprolactone/methacryloylated elastin nanofiber composite membrane and preparation method and application thereof | |
| CN102218160A (en) | Preparation and application of nerve tissue matrix derived tissue engineering scaffold material | |
| Liu et al. | Challenges and opportunities of silk protein hydrogels in biomedical applications | |
| WO2022142350A1 (en) | Injection filling material and preparation process | |
| Kim et al. | Three-dimensional porous collagen/chitosan complex sponge for tissue engineering | |
| CN114904049A (en) | Polycaprolactone microsphere gel containing hyaluronic acid and collagen and preparation method thereof | |
| CN101703807B (en) | Polylactic acid/chitosan composite nano fiber scaffold, preparation method and application thereof | |
| Ma et al. | Injectable hyaluronic acid/poly (γ-glutamic acid) hydrogel with step-by-step tunable properties for soft tissue engineering | |
| CN105176095B (en) | A kind of collagen-based organic silicon rubber porous composite film and preparation method thereof | |
| Cheng et al. | Injectable composite hydrogels encapsulating gelatin methacryloyl/chitosan microspheres as ARPE-19 cell transplantation carriers | |
| CN113248743A (en) | Biocompatible degradable three-dimensional cellulose gel and preparation method and application thereof | |
| Wang et al. | Fabrication, characterization and potential application of biodegradable polydopamine-modified scaffolds based on natural macromolecules | |
| CN110464704B (en) | Preparation method of injectable absorbable anti-infection gel preparation compound | |
| CN101705580A (en) | Preparation method of collagen ultrafine membrane material |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200117 |
|
| RJ01 | Rejection of invention patent application after publication |