CN115192776A - Method for preparing tenacious hydrogel for repairing tendon injury - Google Patents
Method for preparing tenacious hydrogel for repairing tendon injury Download PDFInfo
- Publication number
- CN115192776A CN115192776A CN202210860482.2A CN202210860482A CN115192776A CN 115192776 A CN115192776 A CN 115192776A CN 202210860482 A CN202210860482 A CN 202210860482A CN 115192776 A CN115192776 A CN 115192776A
- Authority
- CN
- China
- Prior art keywords
- solution
- hyaluronic acid
- collagen
- mass
- hydrogel
- 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.)
- Granted
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
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
-
- 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
-
- 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
-
- 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
- A61L27/54—Biologically active materials, e.g. therapeutic substances
-
- 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
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
- A61L2300/414—Growth factors
-
- 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
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
- A61L2300/62—Encapsulated active agents, e.g. emulsified droplets
- A61L2300/622—Microcapsules
-
- 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
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/06—Flowable or injectable implant compositions
-
- 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
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/10—Materials or treatment for tissue regeneration for reconstruction of tendons or ligaments
-
- 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
- C08J2305/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2301/00 or C08J2303/00
- C08J2305/08—Chitin; Chondroitin sulfate; Hyaluronic acid; 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
- C08J2329/00—Characterised by the use of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an alcohol, ether, aldehydo, ketonic, acetal, or ketal radical; Hydrolysed polymers of esters of unsaturated alcohols with saturated carboxylic acids; Derivatives of such polymer
- C08J2329/02—Homopolymers or copolymers of unsaturated alcohols
- C08J2329/04—Polyvinyl alcohol; Partially hydrolysed homopolymers or copolymers of esters of unsaturated alcohols with saturated carboxylic acids
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a method for preparing a tenacious hydrogel for repairing tendon injury, which comprises the following steps: swelling the collagen drug-loaded microspheres, placing the swelled collagen drug-loaded microspheres in a modified hyaluronic acid aqueous solution to obtain a system A, stirring and mixing the system A and a polyvinyl alcohol aqueous solution, and adjusting the pH value to obtain the tenacious hydrogel for repairing tendon injury; the volume of the modified hyaluronic acid aqueous solution is 3-6 times of the mass of the collagen drug-loaded microspheres, the mass of the system A is 1.5-3 times of the mass of the polyvinyl alcohol aqueous solution, and the mass percentage of the polyvinyl alcohol in the polyvinyl alcohol aqueous solution is 15-20 wt%. The tenacious hydrogel for repairing tendon injury can be tightly attached to a tendon injury suture wound, absorbs tissue seepage, carries out drug delivery and promotes the healing of the tendon wound.
Description
Technical Field
The invention belongs to the technical field of hydrogel, and particularly relates to a method for preparing a tough hydrogel for repairing tendon injury.
Background
A tendon is a highly structured fibrous connective tissue for connecting muscles and bones and performing physical activities, and its main function is to transmit power between muscles and bones, stabilize joints, and play a vital role in daily activities. However, when the load on tendons exceeds a critical threshold (as is often the case during strenuous exercise or overuse), tendon tissue is extremely vulnerable to various physical exercises, tendon disease and accidents, and more than 3000 million human tendon transplant surgeries are performed annually worldwide. The hydrogel and the tissue engineering scaffold reported at present can not effectively solve the problem of low cellularity of tendon tissue in the repair process, the repair time is not obviously shortened, and the materials can not be attached to tendon wounds.
The physiological microenvironment of acute/chronic tendon wounds differs from other wounds primarily by the following features: 1. the tendon tissue has low cell content, less blood vessels and low metabolic activity, and the cell proliferation is lower than that of other wounds, so that the tendon recovery after injury is extremely poor; 2. the cells present in the regenerated tendon are not tenocytes (which predominate in healthy tendons), and the composition or arrangement of the ECM is insufficient to meet the mechanical and physiological properties required of this tissue; 3. repair of a tendon rupture results in a reduction in tendon length, which may lead to restricted movement. These defects all result in tendon injuries that are difficult to heal and are easily broken again after breaking. Therefore, starting from repair after tendon injury, developing hydrogels that can promote repair after tendon injury is one of the major challenges in the development of current biomaterials.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for preparing a tough hydrogel for repairing tendon injury aiming at the defects of the prior art. According to the method, collagen drug-loaded microspheres are placed in a modified hyaluronic acid aqueous solution after swelling to obtain a system A, the system A and a polyvinyl alcohol aqueous solution are stirred and mixed, and then the pH value is adjusted to obtain the tough hydrogel for repairing tendon injury, wherein the tough hydrogel for repairing tendon injury can be tightly attached to a tendon injury suture wound, absorbs tissue exudate, carries out drug delivery and promotes the healing of the tendon injury wound.
In order to solve the technical problems, the invention adopts the technical scheme that: a method for preparing a tough hydrogel for repairing tendon injury is characterized by comprising the following steps: swelling the collagen drug-loaded microspheres, placing the swelled collagen drug-loaded microspheres in a modified hyaluronic acid aqueous solution to obtain a system A, stirring and mixing the system A and a polyvinyl alcohol aqueous solution for 1.5-3 min, and adjusting the pH value to obtain the tenacious hydrogel for repairing tendon injury; the volume of the modified hyaluronic acid aqueous solution is 3-6 times of the mass of the collagen drug-loaded microspheres, the unit of the volume of the modified hyaluronic acid aqueous solution is mL, and the unit of the mass of the collagen drug-loaded microspheres is g; the mass percentage of the modified hyaluronic acid in the modified hyaluronic acid aqueous solution is 5-8%; the mass of the system A is 1.5-3 times of that of the polyvinyl alcohol aqueous solution, and the mass percentage of the polyvinyl alcohol in the polyvinyl alcohol aqueous solution is 15-20 wt%.
The method for preparing the tenacious hydrogel for repairing tendon injury is characterized in that the swelling is performed by using PBS (phosphate buffer solution), the volume of the PBS is 3-5 times of the mass of the collagen drug-loaded microspheres, the unit of the volume of the buffer solution is mL, and the unit of the mass of the collagen drug-loaded microspheres is g.
The method for preparing the tenacious hydrogel for repairing tendon injury is characterized in that the pH is adjusted to 7.4.
The method for preparing the tenacious hydrogel for repairing tendon injury is characterized in that the preparation method of the collagen drug-loaded microspheres comprises the following steps:
step 101, placing a growth factor in a collagen solution, and stirring for 0.5-3 hours at the temperature of 37-45 ℃ to obtain a suspension containing the growth factor;
102, stirring and mixing liquid paraffin and an emulsifier for 0.5 to 3 hours at the temperature of 40 to 45 ℃ to obtain an emulsifier-containing system;
step 103, dripping the suspension liquid containing the growth factors in the step 101 into the system containing the emulsifier in the step 102 at the temperature of 37-45 ℃, and stirring for 1-2 hours to obtain an emulsion system containing the growth factors;
step 104, quenching the growth factor-containing emulsion system obtained in the step 103 to 4-10 ℃, and keeping the temperature for 0.5-1 h to obtain a cooled growth factor-containing emulsion system;
105, dropwise adding a glutaraldehyde solution into the cooled growth factor-containing emulsion system in the step 104, stirring for 1.5-3 hours at the temperature of 4-10 ℃ to obtain microspheres, and centrifugally washing, rotary steaming and freeze-drying the microspheres to obtain collagen drug-loaded microspheres;
the method for preparing the tenacious hydrogel for repairing tendon injury is characterized in that in step 101, the mass of the growth factor is 2 multiplied by 10 of the mass of collagen -5 ～8×10 -5 The collagen solution contains 10-20% of collagen by mass, the collagen is pigskin collagen, and the growth factor is a basic fibroblast growth factor.
The method for preparing the tenacious hydrogel for repairing tendon injury is characterized in that in step 102, the emulsifier is Span80, the stirring and mixing speed is 1000-1800 rpm, and the volume of the liquid paraffin is 37-60 times that of the emulsifier.
The method for preparing the tenacious hydrogel for repairing tendon injury is characterized in that in step 105, the volume of the glutaraldehyde solution is 0.1-0.2 times of the volume of the growth factor-containing suspension in step 103, the mass percentage of the glutaraldehyde solution is 10-25 wt%, and the dropping rate is 0.5-3 mL/min.
The method for preparing the tenacious hydrogel for repairing tendon injury is characterized in that the modified hyaluronic acid aqueous solution is a modified hyaluronic acid aqueous solution obtained by mixing modified hyaluronic acid with water, and the preparation method of the modified hyaluronic acid comprises the following steps:
step 201, stirring and activating an acetic acid buffer solution containing a modifier for 0.5-l.5h at the temperature of 18-37 ℃ to obtain an activated solution;
step 202, mixing the activating solution obtained in the step 201 with a hyaluronic acid solution, adjusting the pH to 5.5-6, stirring and reacting for 4-8 hours at the temperature of 18-37 ℃ to obtain a solution containing modified hyaluronic acid, and dialyzing and freeze-drying the solution containing modified hyaluronic acid to obtain the modified hyaluronic acid.
In step 201, the acetic acid buffer solution containing the modifier is prepared by dissolving the modifier in the acetic acid buffer solution, the modifier is 3-aminophenylboronic acid, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, the mass of the 3-aminophenylboronic acid is 3.25% of the mass of the acetic acid buffer solution, and the ratio of the mass of the 3-aminophenylboronic acid, the mass of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and the mass of the N-hydroxysuccinimide is 1: (1-2.5): (1-2.8).
The method for preparing the tenacious hydrogel for repairing tendon injury is characterized in that in step 202, the amount of the 3-aminophenylboronic acid substance is 0.6-1 time of the amount of the hyaluronic acid substance, the hyaluronic acid solution is prepared by mixing hyaluronic acid and 0.5-1% by mass of acetic acid buffer solution, and the mass percentage of the hyaluronic acid in the hyaluronic acid solution is 0.5-1%.
Compared with the prior art, the invention has the following advantages:
1. according to the method, collagen drug-loaded microspheres are placed in a modified hyaluronic acid aqueous solution after being swelled to obtain a system A, the system A and a polyvinyl alcohol aqueous solution are stirred and mixed, and then the pH value is adjusted to obtain the tenacious hydrogel for repairing tendon injury, which can be tightly attached to a tendon injury suture wound, absorb tissue exudate, deliver drugs and promote the healing of the tendon injury wound.
2. The hydrogel prepared by the preparation method is pertinently used for suturing wounds in tendon injury surgical operations, is highly suitable for tendon injury wounds, and meets the toughness requirements of natural tendons.
3. Preferably, the collagen drug-loaded microspheres are prepared by placing the growth factor in a collagen solution, mixing the growth factor with a system of liquid paraffin and an emulsifier, quenching, adding a glutaraldehyde solution, and stirring, so that the collagen drug-loaded microspheres can effectively promote tendon wound healing and meet degradation requirements.
4. Preferably, the hydrogel is prepared by taking 3-aminobenzene boric acid, 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride and N-hydroxysuccinimide as modifiers and mixing and reacting the modified hyaluronic acid and the collagen drug-loaded microspheres with a polyvinyl alcohol aqueous solution, and the plasticity, injectability, biocompatibility and no toxic or side effect of the hydrogel can be realized without a cross-linking agent.
5. The raw materials used in the preparation method of the invention all meet the drug property requirements of FDA, are cheap and easily available, have low cost and are beneficial to popularization and application.
The technical solution of the present invention is further described in detail with reference to the accompanying drawings and embodiments.
Drawings
FIG. 1 shows the modification of hyaluronic acid according to step two of example 1 1 H NMR spectrum.
FIG. 2 is a schematic diagram of the gelling of the tough hydrogel for repairing tendon injury in example 1.
FIG. 3 is a scanning electron microscope image of the tough hydrogel for repairing tendon injury in example 1.
FIG. 4 is a schematic diagram showing the shape adaptability of the tough hydrogel for repairing tendon injury.
Fig. 5 is a schematic diagram of the experimental results of the adhesion strength performance test of the tough hydrogel for repairing tendon injury in example 1.
Fig. 6 is a schematic view of the experimental results of the compressive strain performance test of the tough hydrogel for repairing tendon injury in example 1.
Fig. 7 is a graph showing the results of the strain amplitude scan test of the tough hydrogel for repairing tendon injury in example 1.
Fig. 8 is a schematic diagram showing the results of the self-healing performance test of the tough hydrogel for repairing tendon injury in example 1.
Fig. 9 is a graph showing the results of the cytotoxicity test of the tough hydrogel for repairing tendon injury in example 1.
FIG. 10 is a graph showing the results of the hemolysis rate test of the tough hydrogel for repairing tendon injury in example 1.
Fig. 11 is a schematic diagram showing the results of the test of the cell migration promoting ability of the tough hydrogel for repairing tendon injury in example 1.
FIG. 12 is a photomicrograph of a tendon injury model of a New Zealand white rabbit constructed using the tendon injury repair hydrogel of example 1 and a hydrogel for repairing an implanted tendon injury.
Detailed Description
Example 1
As shown in fig. 1, this embodiment provides a method for preparing a tough hydrogel for tendon injury repair, comprising:
step one, preparing collagen drug-loaded microspheres, which specifically comprises the following steps:
step 101, placing growth factors into a collagen solution, and stirring for 0.5h at the temperature of 37 ℃ to obtain a suspension containing the growth factors; the mass of the growth factor is 2 multiplied by 10 of the mass of the collagen -5 The collagen solution is formed by dissolving collagen in water at the temperature of 37 ℃, and the mass percentage of the collagen in the collagen solution is 15%; the collagen is pigskin collagen, and the growth factor is a basic fibroblast growth factor; the collagen can also be one or more of other natural collagens, recombinant collagens and recombinant collagens doped with natural collagens; the growth factors were purchased from MCE (MedChemExpress), and the collagen was purchased from west ampere macros bio-gene technology, inc;
step 102, stirring and mixing 30mL of liquid paraffin and 0.5mL of emulsifier for 0.5h at the temperature of 40 ℃ to obtain an emulsifier-containing system; the emulsifier is Span80; the stirring and mixing speed is 1000rpm;
step 103, dripping 5mL of the suspension liquid containing the growth factors in the step 101 into the system containing the emulsifier in the step 102 at the temperature of 40 ℃, and stirring for 1h to obtain an emulsion system containing the growth factors; the dropping speed is 1mL/min;
step 104, quenching the growth factor-containing emulsion system obtained in the step 103 to 4 ℃, and keeping the temperature for 0.5h to obtain a cooled growth factor-containing emulsion system;
105, dropwise adding 1mL of glutaraldehyde solution into the cooled growth factor-containing emulsion system obtained in the step 104, stirring for 2 hours at the temperature of 10 ℃ to obtain microspheres, and centrifugally washing, rotationally steaming and freeze-drying the microspheres to obtain collagen drug-loaded microspheres; the mass percentage content of the glutaraldehyde solution is 15wt%; the dropping speed is 1mL/min; the particle size of the collagen drug-loaded microspheres is 8-20 μm; the centrifugal washing is washing in a centrifuge by using isopropanol, ethanol and distilled water alternately, the washing time is 3min each time, and the rotation speed of the centrifuge is 4500rpm;
step two, preparing the modified hyaluronic acid, which specifically comprises the following steps:
step 201, stirring and activating an acetic acid buffer solution containing a modifier for lh at the temperature of 25 ℃ to obtain an activated solution; the pH value of the acetic acid buffer solution containing the modifier is 5.8; the acetic acid buffer solution containing the modifier is prepared by dissolving the modifier in 40mL of acetic acid buffer solution, the modifier is 3-aminobenzene boric acid, 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride and N-hydroxysuccinimide, the mass of the 3-aminobenzene boric acid is 3.25% of that of the acetic acid buffer solution, and the mass ratio of the 3-aminobenzene boric acid, the 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride and the N-hydroxysuccinimide is 1:2:2;
step 202, mixing the activating solution obtained in the step 201 with a hyaluronic acid solution, adjusting the pH to 5.5, stirring and reacting for 4 hours at the temperature of 25 ℃ to obtain a solution containing modified hyaluronic acid, putting the solution containing modified hyaluronic acid into a dialysis bag for dialysis for 3 days, changing water for 5 times every day, and freeze-drying to obtain the modified hyaluronic acid; the amount of the 3-aminophenylboronic acid substance is 0.7 times the amount of the substance of the hyaluronic acid; the hyaluronic acid solution is prepared by mixing hyaluronic acid and 0.8% of acetic acid buffer solution in percentage by mass, and the percentage by mass of hyaluronic acid in the hyaluronic acid solution is 1%; the dialyzing agent for dialysis is distilled water, and the cut-off molecular weight of the dialysis bag is 8000;
and step three, stirring a system containing the collagen drug-loaded microspheres and the modified hyaluronic acid and a polyvinyl alcohol aqueous solution for reaction to obtain the tenacious hydrogel for repairing tendon injury, which specifically comprises the following steps:
step 301, irradiating 0.lg of the collagen drug-loaded microspheres obtained in the step one by Co60 for sterilization to obtain sterilized collagen drug-loaded microspheres;
step 302,Mixing the sterilized collagen drug-loaded microspheres obtained in the step 301 with 0.5mL of buffer solution, and stirring for 3min to obtain swollen collagen drug-loaded microspheres; the buffer solution is PBS solution, and the PBS solution can be composed of 8g NaCl, 0.2g KCI and 1.44g Na 2 HPO 4 And 0.24g KH 2 PO 4 Dissolving in 800ml distilled water to prepare PBS solution;
step 303, mixing the modified hyaluronic acid obtained in the step two with water to obtain a modified hyaluronic acid aqueous solution with the mass percentage of 5%;
304, placing the swelled collagen drug-loaded microspheres obtained in the step 302 in 0.6mL of the modified hyaluronic acid aqueous solution obtained in the step 303 to obtain a system A;
step 305, dissolving polyvinyl alcohol in water to obtain a polyvinyl alcohol aqueous solution with the polyvinyl alcohol content of 20wt%; the molecular weight of the polyvinyl alcohol is 89000-98000;
step 306, stirring and mixing the system A in the step 304 and the polyvinyl alcohol aqueous solution in the step 305 for 2min to obtain a system B; the mass of the system A is 3 times of that of the polyvinyl alcohol aqueous solution;
and 307, adjusting the pH value of the system B in the step 306 to 7.4 to obtain the tenacious hydrogel for repairing the tendon injury.
Example 2
This example provides a method for preparing a tough hydrogel for repairing tendon injury, comprising:
step one, preparing collagen drug-loaded microspheres, which specifically comprises the following steps:
step 101, placing a growth factor in a collagen solution, and stirring for 2 hours at the temperature of 37 ℃ to obtain a suspension containing the growth factor; the mass of the growth factor is 4 multiplied by 10 of the mass of the collagen -5 The collagen solution is formed by dissolving collagen in water at the temperature of 37 ℃, and the mass percentage of the collagen in the collagen solution is 10%; the collagen is pigskin collagen, and the growth factor is basic fibroblast growth factor; the collagen can also be other natural collagen,One or more of recombinant collagen and recombinant collagen doped with natural collagen;
step 102, stirring and mixing 30mL of liquid paraffin and 0.8mL of emulsifier for 1h at the temperature of 40 ℃ to obtain an emulsifier-containing system; the emulsifier is Span80; the stirring and mixing speed is 1800rpm;
step 103, dripping 5mL of the suspension liquid containing the growth factors in the step 101 into the system containing the emulsifier in the step 102 at the temperature of 40 ℃, and stirring for 1.5h to obtain an emulsion system containing the growth factors; the dropping rate is 0.5mL/min;
step 104, quenching the growth factor-containing emulsion system obtained in the step 103 to 10 ℃, and keeping the temperature for 0.5h to obtain a cooled growth factor-containing emulsion system;
105, dropwise adding 1mL of glutaraldehyde solution into the cooled growth factor-containing emulsion system obtained in the step 104, stirring for 1.5 hours at the temperature of 10 ℃ to obtain microspheres, and performing centrifugal washing, rotary evaporation and freeze drying on the microspheres to obtain collagen drug-loaded microspheres; the mass percentage content of the glutaraldehyde solution is 10wt%; the dropping rate is 0.5mL/min; the particle size of the collagen drug-loaded microspheres is 8-20 μm; the centrifugal washing is washing in a centrifuge by using isopropanol, ethanol and distilled water alternately, the washing time is 3min each time, and the rotating speed of the centrifuge is 4500rpm;
step two, preparing the modified hyaluronic acid, which specifically comprises the following steps:
step 201, stirring and activating an acetic acid buffer solution containing a modifier for lh at the temperature of 20 ℃ to obtain an activated solution; the pH value of the acetic acid buffer solution containing the modifier is 5.8; the acetic acid buffer solution containing the modifier is prepared by dissolving the modifier in 40mL of acetic acid buffer solution, the modifier is 3-aminobenzene boric acid, 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride and N-hydroxysuccinimide, the mass of the 3-aminobenzene boric acid is 3.25% of that of the acetic acid buffer solution, and the mass ratio of the 3-aminobenzene boric acid, the 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride and the N-hydroxysuccinimide is 1:2.5: 2.8 of;
step 202, mixing the activating solution obtained in the step 201 with a hyaluronic acid solution, adjusting the pH to 5.5, stirring and reacting for 6 hours at the temperature of 20 ℃ to obtain a solution containing modified hyaluronic acid, putting the solution containing modified hyaluronic acid into a dialysis bag for dialysis for 3 days, changing water for 5 times every day, and freeze-drying to obtain the modified hyaluronic acid; the amount of the 3-aminophenylboronic acid substance is 0.6 times the amount of the substance of the hyaluronic acid; the hyaluronic acid solution is prepared by mixing hyaluronic acid and 0.8% of acetic acid buffer solution in percentage by mass, and the percentage by mass of hyaluronic acid in the hyaluronic acid solution is 1%; the dialyzing agent for dialysis is distilled water, and the cut-off molecular weight of the dialysis bag is 8000;
and step three, stirring a system containing the collagen drug-loaded microspheres and the modified hyaluronic acid and a polyvinyl alcohol aqueous solution for reaction to obtain the tenacious hydrogel for repairing tendon injury, which specifically comprises the following steps:
301, irradiating 0.l5g of the collagen drug-loaded microspheres obtained in the first step by Co60 for sterilization to obtain sterilized collagen drug-loaded microspheres;
step 302, mixing the sterilized collagen drug-loaded microspheres obtained in step 301 with 0.5mL of buffer solution, and stirring for 1min to obtain swollen collagen drug-loaded microspheres; the buffer solution is PBS solution, and the PBS solution can be composed of 8g NaCl, 0.2g KCI and 1.44g Na 2 HPO 4 And 0.24g KH 2 PO 4 Dissolving in 800ml distilled water to prepare PBS solution;
step 303, mixing the modified hyaluronic acid obtained in the step two with water to obtain a modified hyaluronic acid aqueous solution with the mass percentage of 5%;
304, placing the swelled collagen drug-loaded microspheres obtained in the step 302 in 0.6mL of the modified hyaluronic acid aqueous solution obtained in the step 303 to obtain a system A;
305, dissolving polyvinyl alcohol in water to obtain a polyvinyl alcohol aqueous solution with the polyvinyl alcohol mass percentage of 20wt%; the molecular weight of the polyvinyl alcohol is 89000-98000;
step 306, stirring and mixing the system A in the step 304 and the polyvinyl alcohol aqueous solution in the step 305 for 1.5min to obtain a system B; the mass of the system A is 1.5 times of that of the polyvinyl alcohol aqueous solution;
and 307, adjusting the pH value of the system B in the step 306 to 7.4 to obtain the tenacious hydrogel for repairing the tendon injury.
The performance of the tough hydrogel for repairing tendon injury of this example was substantially the same as that of example 1.
Example 3
The embodiment provides a method for preparing a tough hydrogel for repairing tendon injury, which comprises the following steps:
step one, preparing collagen drug-loaded microspheres, which specifically comprises the following steps:
step 101, placing growth factors into a collagen solution, and stirring for 3 hours at the temperature of 37 ℃ to obtain a suspension containing the growth factors; the quality of the growth factor is 6 multiplied by 10 of the quality of the collagen -5 The collagen solution is formed by dissolving collagen in water at 37 ℃, and the mass percentage of the collagen in the collagen solution is 20%; the collagen is pigskin collagen, and the growth factor is basic fibroblast growth factor; the collagen can also be one or more of other natural collagens, recombinant collagens and recombinant collagens doped with natural collagens;
step 102, stirring and mixing 30mL of liquid paraffin and 0.6mL of emulsifier for 3h at the temperature of 40 ℃ to obtain an emulsifier-containing system; the emulsifier is Span80; the stirring and mixing speed is 1000rpm;
step 103, dripping 5mL of the suspension liquid containing the growth factors in the step 101 into the system containing the emulsifier in the step 102 at the temperature of 45 ℃, and stirring for 1h to obtain an emulsion system containing the growth factors; the dropping rate is 1.5mL/min;
step 104, quenching the growth factor-containing emulsion system obtained in the step 103 to 6 ℃, and keeping the temperature for 1h to obtain a cooled growth factor-containing emulsion system;
105, dropwise adding 1mL of glutaraldehyde solution into the cooled growth factor-containing emulsion system obtained in the step 104, stirring for 2 hours at the temperature of 6 ℃ to obtain microspheres, and performing centrifugal washing, rotary evaporation and freeze drying on the microspheres to obtain collagen drug-loaded microspheres; the mass percentage content of the glutaraldehyde solution is 20wt%; the dropping rate is 1.5mL/min; the particle size of the collagen drug-loaded microspheres is 8-20 μm; the centrifugal washing is washing in a centrifuge by using isopropanol, ethanol and distilled water alternately, the washing time is 3min each time, and the rotating speed of the centrifuge is 4500rpm;
step two, preparing the modified hyaluronic acid, which specifically comprises the following steps:
step 201, stirring and activating a modifier-containing acetic acid buffer solution l h at the temperature of 25 ℃ to obtain an activation solution; the pH value of the acetic acid buffer solution containing the modifier is 5.8; the acetic acid buffer solution containing the modifier is prepared by dissolving the modifier in 40mL of acetic acid buffer solution, the modifier is 3-aminobenzene boric acid, 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride and N-hydroxysuccinimide, the mass of the 3-aminobenzene boric acid is 3.25% of that of the acetic acid buffer solution, and the mass ratio of the 3-aminobenzene boric acid, the 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride and the N-hydroxysuccinimide is 1:2:2;
step 202, mixing the activating solution obtained in the step 201 with a hyaluronic acid solution, adjusting the pH to 5.5, stirring and reacting for 8 hours at the temperature of 18 ℃ to obtain a solution containing modified hyaluronic acid, placing the solution containing modified hyaluronic acid in a dialysis bag for dialysis for 3 days, changing water for 5 times every day, and freeze-drying to obtain the modified hyaluronic acid; the amount of the 3-aminophenylboronic acid substance is 0.9 times the amount of the substance of the hyaluronic acid; the hyaluronic acid solution is prepared by mixing hyaluronic acid and 1% acetic acid buffer solution by mass, and the mass percentage of hyaluronic acid in the hyaluronic acid solution is 0.5%; the dialyzing agent for dialysis is distilled water, and the cut-off molecular weight of the dialysis bag is 8000;
and step three, stirring a system containing the collagen drug-loaded microspheres and the modified hyaluronic acid and a polyvinyl alcohol aqueous solution for reaction to obtain the tenacious hydrogel for repairing tendon injury, which specifically comprises the following steps:
step 301, irradiating and sterilizing 0.l5g of the collagen drug-loaded microspheres obtained in the step one by Co60 to obtain sterilized collagen drug-loaded microspheres;
step 302, mixing the sterilized collagen drug-loaded microspheres obtained in step 301 with 0.5mL of buffer solution, and stirring for 6min to obtain swollen collagen drug-loaded microspheres; the buffer solution is PBS solution, and the PBS solution can be composed of 8g NaCl, 0.2g KCI and 1.44g Na 2 HPO 4 And 0.24g KH 2 PO 4 Dissolving in 800ml distilled water to prepare PBS solution;
step 303, mixing the modified hyaluronic acid obtained in the step two with water to obtain a modified hyaluronic acid aqueous solution with the mass percentage of 8%;
304, placing the swelled collagen drug-loaded microspheres obtained in the step 302 in 0.6mL of the modified hyaluronic acid aqueous solution obtained in the step 303 to obtain a system A;
305, dissolving polyvinyl alcohol in water to obtain a polyvinyl alcohol aqueous solution with the polyvinyl alcohol mass percentage of 20wt%; the molecular weight of the polyvinyl alcohol is 89000-98000;
step 306, stirring and mixing the system A obtained in the step 304 and the polyvinyl alcohol aqueous solution obtained in the step 305 for 3min to obtain a system B; the mass of the system A is 2 times of that of the polyvinyl alcohol aqueous solution;
and 307, adjusting the pH value of the system B in the step 306 to 7.4 to obtain the tenacious hydrogel for repairing the tendon injury.
The performance of the tough hydrogel for repairing tendon injury of this example was substantially the same as that of example 1.
Example 4
This example provides a method for preparing a tough hydrogel for repairing tendon injury, comprising:
step one, preparing collagen drug-loaded microspheres, which specifically comprises the following steps:
step 101, placing growth factors in a collagen solution at a temperature of 45 DEG CStirring for 2h to obtain suspension containing growth factors; the quality of the growth factor is 8 multiplied by 10 of the quality of the collagen -5 The collagen solution is formed by dissolving collagen in water at the temperature of 45 ℃, and the mass percentage of the collagen in the collagen solution is 10%; the collagen is pigskin collagen, and the growth factor is a basic fibroblast growth factor; the collagen can also be one or more of other natural collagens, recombinant collagens and recombinant collagens doped with natural collagens;
step 102, stirring and mixing 30mL of liquid paraffin and 0.5mL of emulsifier for 1h at the temperature of 40 ℃ to obtain an emulsifier-containing system; the emulsifier is Span80; the stirring and mixing speed is 1200rpm;
step 103, dripping 5mL of the suspension liquid containing the growth factors in the step 101 into the system containing the emulsifier in the step 102 at 37 ℃, and stirring for 2 hours to obtain an emulsion system containing the growth factors; the dropping speed is 2mL/min;
step 104, quenching the growth factor-containing emulsion system obtained in the step 103 to 7 ℃, and keeping the temperature for 1h to obtain a cooled growth factor-containing emulsion system;
105, dropwise adding 0.5mL of glutaraldehyde solution into the cooled growth factor-containing emulsion system obtained in the step 104, stirring for 3 hours at the temperature of 7 ℃ to obtain microspheres, and performing centrifugal washing, rotary evaporation and freeze drying on the microspheres to obtain collagen drug-loaded microspheres; the mass percentage content of the glutaraldehyde solution is 25wt%; the dropping speed is 3mL/min; the particle size of the collagen drug-loaded microspheres is 8-20 μm; the centrifugal washing is washing in a centrifuge by using isopropanol, ethanol and distilled water alternately, the washing time is 3min each time, and the rotation speed of the centrifuge is 4500rpm;
step two, preparing the modified hyaluronic acid, which specifically comprises the following steps:
step 201, stirring and activating an acetic acid buffer solution containing a modifier by 0.5h at the temperature of 18 ℃ to obtain an activated solution; the pH value of the acetic acid buffer solution containing the modifier is 5.8; the acetic acid buffer solution containing the modifier is prepared by dissolving the modifier in 40mL of acetic acid buffer solution, the modifier is 3-aminobenzene boric acid, 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride and N-hydroxysuccinimide, the mass of the 3-aminobenzene boric acid is 3.25% of that of the acetic acid buffer solution, and the mass ratio of the 3-aminobenzene boric acid, the 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride and the N-hydroxysuccinimide is 1: 1.5:1.8;
step 202, mixing the activating solution obtained in the step 201 with a hyaluronic acid solution, adjusting the pH to 5.5, stirring and reacting for 8 hours at the temperature of 37 ℃ to obtain a solution containing modified hyaluronic acid, putting the solution containing modified hyaluronic acid into a dialysis bag for dialysis for 3 days, changing water for 5 times every day, and freeze-drying to obtain the modified hyaluronic acid; the amount of the 3-aminophenylboronic acid substance is 1 time of the amount of the substance of the hyaluronic acid; the hyaluronic acid solution is prepared by mixing hyaluronic acid and 0.5% by mass of acetic acid buffer solution, and the mass percentage of hyaluronic acid in the hyaluronic acid solution is 0.5%; the dialyzing agent for dialysis is distilled water, and the cut-off molecular weight of the dialysis bag is 8000;
and step three, stirring a system containing the collagen drug-loaded microspheres and the modified hyaluronic acid and a polyvinyl alcohol aqueous solution for reaction to obtain the tenacious hydrogel for repairing tendon injury, which specifically comprises the following steps:
step 301, irradiating 0.2g of the collagen drug-loaded microspheres obtained in the step one by Co60 for sterilization to obtain sterilized collagen drug-loaded microspheres;
step 302, mixing the sterilized collagen drug-loaded microspheres obtained in step 301 with 0.8mL of buffer solution, and stirring for 3min to obtain swollen collagen drug-loaded microspheres; the buffer solution is PBS solution, and the PBS solution can be composed of 8g NaCl, 0.2g KCI and 1.44g Na 2 HPO 4 And 0.24g KH 2 PO 4 Dissolving in 800ml distilled water to prepare PBS solution;
step 303, mixing the modified hyaluronic acid obtained in the step two with water to obtain a modified hyaluronic acid aqueous solution with the mass percentage of 8%;
304, placing the swelled collagen drug-loaded microspheres obtained in the step 302 in 0.6mL of the modified hyaluronic acid aqueous solution obtained in the step 303 to obtain a system A;
305, dissolving polyvinyl alcohol in water to obtain a polyvinyl alcohol aqueous solution with the polyvinyl alcohol mass percentage of 15wt%; the molecular weight of the polyvinyl alcohol is 89000-98000;
step 306, stirring and mixing the system A in the step 304 and the polyvinyl alcohol aqueous solution in the step 305 for 2min to obtain a system B; the mass of the system A is 1.5 times of that of the polyvinyl alcohol aqueous solution;
and 307, adjusting the pH value of the system B in the step 306 to 7.4 to obtain the tenacious hydrogel for repairing the tendon injury.
The performance of the tough hydrogel for repairing tendon injury of the present example is substantially the same as that of example 1.
Example 5
This example provides a method for preparing a tough hydrogel for repairing tendon injury, comprising:
step one, preparing collagen drug-loaded microspheres, which specifically comprises the following steps:
step 101, placing growth factors into a collagen solution, and stirring for 0.5h at the temperature of 37 ℃ to obtain a suspension containing the growth factors; the quality of the growth factor is 2 multiplied by 10 of the quality of the collagen -5 The collagen solution is formed by dissolving collagen in water at the temperature of 37 ℃, and the mass percentage of the collagen in the collagen solution is 20%; the collagen is pigskin collagen, and the growth factor is basic fibroblast growth factor; the collagen can also be one or more of other natural collagens, recombinant collagens and recombinant collagens doped with natural collagens;
step 102, stirring and mixing 30mL of liquid paraffin and 0.8mL of emulsifier for 1.5h at the temperature of 45 ℃ to obtain an emulsifier-containing system; the emulsifier is Span80; the stirring and mixing speed is 1200rpm;
step 103, dripping 10mL of the suspension liquid containing the growth factors in the step 101 into the system containing the emulsifier in the step 102 at the temperature of 40 ℃, and stirring for 0.5h to obtain an emulsion system containing the growth factors; the dropping rate is 1.8mL/min;
step 104, quenching the growth factor-containing emulsion system obtained in the step 103 to 4 ℃, and keeping the temperature for 0.5h to obtain a cooled growth factor-containing emulsion system;
105, dropwise adding 1mL of glutaraldehyde solution into the cooled growth factor-containing emulsion system obtained in the step 104, stirring for 3 hours at the temperature of 4 ℃ to obtain microspheres, and centrifugally washing, rotationally steaming and freeze-drying the microspheres to obtain collagen drug-loaded microspheres; the mass percentage content of the glutaraldehyde solution is 10wt%; the dropping rate is 0.8mL/min; the particle size of the collagen drug-loaded microspheres is 8-20 μm; the centrifugal washing is washing in a centrifuge by using isopropanol, ethanol and distilled water alternately, the washing time is 3min each time, and the rotating speed of the centrifuge is 4500rpm;
step two, preparing the modified hyaluronic acid, which specifically comprises the following steps:
step 201, stirring and activating an acetic acid buffer solution containing a modifier for l.5h at the temperature of 37 ℃ to obtain an activation solution; the pH value of the acetic acid buffer solution containing the modifier is 5.8; the acetic acid buffer solution containing the modifier is prepared by dissolving the modifier in 40mL of acetic acid buffer solution, the modifier is 3-aminobenzene boric acid, 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride and N-hydroxysuccinimide, the mass of the 3-aminobenzene boric acid is 3.25% of that of the acetic acid buffer solution, and the mass ratio of the 3-aminobenzene boric acid, the 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride and the N-hydroxysuccinimide is 1: 1:1;
step 202, mixing the activating solution obtained in the step 201 with a hyaluronic acid solution, adjusting the pH to 6.0, stirring and reacting for 6 hours at the temperature of 35 ℃ to obtain a solution containing modified hyaluronic acid, putting the solution containing modified hyaluronic acid into a dialysis bag for dialysis for 3 days, changing water for 5 times every day, and freeze-drying to obtain the modified hyaluronic acid; the amount of the 3-aminophenylboronic acid substance is 0.9 times the amount of the substance of the hyaluronic acid; the hyaluronic acid solution is prepared by mixing hyaluronic acid and 0.5% acetic acid buffer solution in percentage by mass, and the percentage by mass of hyaluronic acid in the hyaluronic acid solution is 1%; the dialyzing agent for dialysis is distilled water, and the cut-off molecular weight of the dialysis bag is 8000;
and step three, stirring a system containing the collagen drug-loaded microspheres and the modified hyaluronic acid and a polyvinyl alcohol aqueous solution for reaction to obtain the tenacious hydrogel for repairing tendon injury, which specifically comprises the following steps:
step 301, irradiating 0.lg of the collagen drug-loaded microspheres obtained in the step one by Co60 for sterilization to obtain sterilized collagen drug-loaded microspheres;
step 302, mixing the sterilized collagen drug-loaded microspheres obtained in step 301 with 0.5mL of buffer solution, and stirring for 6min to obtain swollen collagen drug-loaded microspheres; the buffer solution is PBS solution, and the PBS solution can be composed of 8g NaCl, 0.2g KCI, 1.44g Na 2 HPO 4 And 0.24g KH 2 PO 4 Dissolving in 800ml distilled water to prepare PBS solution;
step 303, mixing the modified hyaluronic acid obtained in the step two with water to obtain a modified hyaluronic acid aqueous solution with the mass percentage of 5%;
304, placing the swelled collagen drug-loaded microspheres obtained in the step 302 in 0.6mL of the modified hyaluronic acid aqueous solution obtained in the step 303 to obtain a system A;
305, dissolving polyvinyl alcohol in water to obtain a polyvinyl alcohol aqueous solution with the polyvinyl alcohol mass percentage of 15wt%; the molecular weight of the polyvinyl alcohol is 89000-98000;
step 306, stirring and mixing the system A in the step 304 and the polyvinyl alcohol aqueous solution in the step 305 for 1.5min to obtain a system B; the mass of the system A is 2 times of that of the polyvinyl alcohol aqueous solution;
and 307, adjusting the pH value of the system B in the step 306 to 7.4 to obtain the tenacious hydrogel for repairing the tendon injury.
The performance of the tough hydrogel for repairing tendon injury of this example was substantially the same as that of example 1.
Performance testing
FIG. 1 shows the modification of hyaluronic acid according to step two of example 1 1 H NMR spectrum. According to the figure 1, the peaks at 7.4, 7.8 and 8.2ppm are characteristic peaks of phenyl protons, and the peak at 1.9ppm is characteristic peak of sodium hyaluronate, which shows that the modifier 3-aminophenylboronic acid successfully modifies hyaluronic acid.
FIG. 2 is a schematic diagram of the gelling of the tough hydrogel for repairing tendon injury in example 1. As can be seen from FIG. 2, the method of the present invention can form gel rapidly in a short time, and the gel forming condition is good.
FIG. 3 is a scanning electron microscope image of the tough hydrogel for repairing tendon injury in example 1. As can be seen from FIG. 3, the tenacious hydrogel for repairing tendon injury of the present invention has a uniform porous structure with a pore size of about 5-30 μm.
As can be seen from the comprehensive graphs of FIGS. 1 to 3, the tenacious hydrogel for repairing tendon injury successfully prepared by the method of the invention.
FIG. 4 is a schematic diagram showing the shape adaptability of the tough hydrogel for repairing tendon injury. As can be seen from FIG. 4, the hydrogel can slowly flow under the action of gravity to adapt to tendon injuries of different shapes, which indicates that the tough hydrogel for repairing tendon injuries of the present invention has adaptivity.
Fig. 5 is a schematic diagram showing the experimental results of the adhesion strength performance test of the tough hydrogel for repairing tendon injury in example 1, wherein the test method is a pig skin lap shear experiment, and two pieces of pig skin are tightly bonded together by using the hydrogel (an adhesion interface is 2.0cm × 2.0 cm). As can be seen from FIG. 5, the hydrogel had an adhesive strength of 41.25kPa at 25 ℃, indicating that the tenacious hydrogel for repairing tendon injuries of the present invention had excellent tissue adhesion.
Fig. 6 is a schematic diagram of the experimental results of the compressive strain performance test of the tough hydrogel for repairing tendon injury in example 1, wherein the compressive strain performance test is performed by a universal material compressor, and the test method comprises the following steps: firstly, the hydrogel is placed in a cylindrical mold with the height of 10mm and the diameter of 10mm for shaping for 10min, taken out, placed on a workbench, set the compression strain of a compressor to be 90% and the speed to be 1mm/min, and recorded with the compression displacement and the compression stress, and the result is shown in fig. 6. As can be seen from FIG. 6, the compressive strain of the hydrogel at 25 ℃ was 27.05kPa, indicating that the strong and tough hydrogel for repairing tendon injury of the present invention has excellent compressive properties.
Fig. 7 is a schematic diagram showing the results of the scanning test of the strain amplitude of the tough hydrogel for repairing tendon injury in example 1, wherein the strain amplitude is tested by a rotational rheometer at 25 ℃. As can be seen from FIG. 7, the hydrogel collapsed at 25 ℃ when the strain was 300%, indicating that the tough hydrogel for repairing tendon injury of the present invention has excellent tensile properties.
Fig. 8 is a schematic diagram of a self-healing performance test result of the tough hydrogel for repairing tendon injury in example 1, and it can be seen from fig. 8 that the hydrogel can rapidly self-heal after being damaged at 25 ℃, and mechanical properties can be restored after self-healing, indicating that the tough hydrogel for repairing tendon injury has rapid self-healing performance.
It can be seen from fig. 4 to 8 that the tough hydrogel for repairing tendon injury of the present invention can be tightly attached to a wound after tendon injury suture in clinical application to promote tendon repair.
Fig. 9 is a graph showing the results of the cytotoxicity test of the tough hydrogel for repairing tendon injury in example 1. The test method comprises soaking the hydrogel in RPM1-1640 medium at 37 deg.C for 72 hr to obtain 0.1g/mL hydrogel extract, replacing the culture medium of mouse fibroblast (L929 cell) after normal culture in RPMI-1640 medium for 24 hr with the hydrogel extract, culturing for 24 hr, 48 hr and 72 hr, measuring absorbance by MTT method, calculating the survival rate of L929 cell, and performing cell dying staining by AO-EB kit, each test repeating 6 parallel groups. As can be seen from FIG. 9, the cell survival rates at 24 hours, 48 hours and 72 hours are all above 85%, which indicates that the tenacious hydrogel for repairing tendon injury of the present invention has good biocompatibility, safety and no toxicity.
FIG. 10 is a graph showing the results of the hemolysis rate test of the tough hydrogel for repairing tendon injury in example 1. The test method comprises soaking the hydrogel in physiological saline at 37 ℃ for 72 hours to obtain a leaching solution with a concentration of 0.1g/mL, collecting fresh blood (5 mL) from ear veins of New Zealand white rabbits, separating red blood cells at 2000rpm for 5min, washing the separated red blood cells with physiological saline until the supernatant is colorless and transparent, diluting the red blood cells to a final concentration of 2% (v/v), mixing 0.5mL of the leaching solution and an equal amount of 2% (v/v) RBC suspension in physiological saline in a 1mL centrifuge tube, culturing the mixture at 37 ℃ for 1h, and measuring the absorbance of the supernatant at 545nm after centrifugation, wherein physiological saline and deionized water are used as negative and positive controls, respectively, and then evaluating the hemolysis rate below 5% by an in vitro hemolysis test, which is considered as no hemolysis. As can be seen from FIG. 10, the hemolysis rates of the hydrogels were all below 5%, indicating that the tough hydrogel for repairing tendon injuries of the present invention does not cause erythrocyte rupture and has good blood compatibility.
FIG. 11 is a graph showing the results of the test of cell migration promoting ability of the tough hydrogel for tendon injury repair of example 1, which comprises culturing L929 cells in a six-well plate with RPMI-1640 medium for 24 hours, aspirating the medium, scratching the cell layer with 200. Mu.L pipette tip, further culturing the cells with hydrogel leaching solution with concentration of 0.1g/mL, culturing the cells for 12 and 24 hours, and observing the scratch closure. According to fig. 11, the cell mobility of the hydrogel group is increased by 53.28 ± 5.34% in 24 hours, which is significantly different from that of the control group, indicating that the hydrogel can effectively promote wound healing.
Fig. 12 is a macroscopic photograph of a tendon injury model of a new zealand rabbit constructed using the tough hydrogel for repairing tendon injury of example 1 and a tough hydrogel for repairing implanted tendon injury, and it can be seen from fig. 12 that the hydrogel can be tightly attached to a tendon injury site and can be filled into a tendon injury suture site to more effectively promote healing of tendon injury.
As can be seen from FIGS. 9 to 12, the tenacious hydrogel for repairing tendon injury of the present invention has excellent biocompatibility.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and any simple modifications, changes and equivalent structural changes made to the above embodiment according to the technical spirit of the present invention still fall within the protection scope of the technical solution of the present invention.
Claims (10)
1. A method for preparing a tough hydrogel for repairing tendon injury is characterized by comprising the following steps: swelling the collagen drug-loaded microspheres, placing the swelled collagen drug-loaded microspheres in a modified hyaluronic acid aqueous solution to obtain a system A, stirring and mixing the system A and a polyvinyl alcohol aqueous solution for 1.5-3 min, and adjusting the pH value to obtain the tenacious hydrogel for repairing tendon injury; the volume of the modified hyaluronic acid aqueous solution is 3-6 times of the mass of the collagen drug-loaded microspheres, the unit of the volume of the modified hyaluronic acid aqueous solution is mL, and the unit of the mass of the collagen drug-loaded microspheres is g; the mass percentage of the modified hyaluronic acid in the modified hyaluronic acid aqueous solution is 5-8%; the mass of the system A is 1.5-3 times of that of the polyvinyl alcohol aqueous solution, and the mass percentage of the polyvinyl alcohol in the polyvinyl alcohol aqueous solution is 15-20 wt%.
2. The method for preparing the tenacious hydrogel for repairing tendon injury according to claim 1, wherein the swelling is performed with PBS (phosphate buffered saline) solution, the volume of the PBS solution is 3-5 times of the mass of the collagen drug-loaded microspheres, the volume of the buffer solution is in mL, and the mass of the collagen drug-loaded microspheres is in g.
3. The method for preparing a tough hydrogel for tendon injury repair according to claim 1 wherein the pH is adjusted to pH 7.4.
4. The method for preparing the tenacious hydrogel for repairing tendon injury according to claim 1, wherein the preparation method of the collagen drug-loaded microspheres comprises the following steps:
step 101, placing a growth factor in a collagen solution, and stirring for 0.5-3 hours at the temperature of 37-45 ℃ to obtain a suspension containing the growth factor;
102, stirring and mixing liquid paraffin and an emulsifier for 0.5 to 3 hours at the temperature of 40 to 45 ℃ to obtain an emulsifier-containing system;
step 103, dripping the suspension liquid containing the growth factors in the step 101 into the system containing the emulsifier in the step 102 at the temperature of 37-45 ℃, and stirring for 1-2 hours to obtain an emulsion system containing the growth factors;
step 104, quenching the growth factor-containing emulsion system obtained in the step 103 to 4-10 ℃, and keeping the temperature for 0.5-1 h to obtain a cooled growth factor-containing emulsion system;
and 105, dropwise adding a glutaraldehyde solution into the cooled growth factor-containing emulsion system obtained in the step 104, stirring for 1.5-3 hours at the temperature of 4-10 ℃ to obtain microspheres, and performing centrifugal washing, rotary evaporation and freeze drying on the microspheres to obtain the collagen drug-loaded microspheres.
5. The method for preparing a tough hydrogel for repairing tendon injury as claimed in claim 4, wherein in step 101, the mass of said growth factor is 2 x 10 of the mass of collagen -5 ～8×10 -5 The collagen solution contains 10-20% of collagen by mass, the collagen is pigskin collagen, and the growth factor is a basic fibroblast growth factor.
6. The method for preparing the tough hydrogel for repairing tendon injury according to claim 4, wherein in step 102, the emulsifier is Span80, the stirring and mixing speed is 1000-1800 rpm, and the volume of the liquid paraffin is 37-60 times that of the emulsifier.
7. The method for preparing the tenacious hydrogel for repairing tendon injury according to claim 4, wherein in step 105, the volume of the glutaraldehyde solution is 0.1-0.2 times of the volume of the growth factor-containing suspension in step 103, the mass percentage of the glutaraldehyde solution is 10-25 wt%, and the dropping rate is 0.5-3 mL/min.
8. The method for preparing the tenacious hydrogel for tendon injury repair according to claim 1, wherein the aqueous solution of modified hyaluronic acid is an aqueous solution of modified hyaluronic acid obtained by mixing modified hyaluronic acid with water, and the method for preparing the modified hyaluronic acid comprises:
step 201, stirring and activating an acetic acid buffer solution containing a modifier for 0.5-l.5h at the temperature of 18-37 ℃ to obtain an activated solution;
step 202, mixing the activating solution obtained in the step 201 with a hyaluronic acid solution, adjusting the pH to 5.5-6, stirring and reacting at the temperature of 18-37 ℃ for 4-8 hours to obtain a solution containing modified hyaluronic acid, and dialyzing and freeze-drying the solution containing modified hyaluronic acid to obtain the modified hyaluronic acid.
9. The method for preparing the tough hydrogel for repairing tendon injury according to claim 8, wherein in step 201, the acetic acid buffer solution containing the modifying agent is prepared by dissolving the modifying agent in the acetic acid buffer solution, the modifying agent is 3-aminobenzeneboronic acid, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, the mass of the 3-aminobenzeneboronic acid is 3.25% of the mass of the acetic acid buffer solution, and the ratio of the mass of the 3-aminobenzeneboronic acid, the mass of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and the mass of the N-hydroxysuccinimide is 1: (1-2.5): (1-2.8).
10. The method for preparing the tenacious hydrogel for repairing tendon injury according to claim 8, wherein in step 202, the amount of the 3-aminophenylboronic acid is 0.6 to 1 times of the amount of the substance of the hyaluronic acid, the hyaluronic acid solution is prepared by mixing the hyaluronic acid with 0.5 to 1% by mass of acetic acid buffer solution, and the mass percentage of the hyaluronic acid in the hyaluronic acid solution is 0.5 to 1%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210860482.2A CN115192776B (en) | 2022-07-21 | 2022-07-21 | Method for preparing tough hydrogel for repairing tendon injury |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210860482.2A CN115192776B (en) | 2022-07-21 | 2022-07-21 | Method for preparing tough hydrogel for repairing tendon injury |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115192776A true CN115192776A (en) | 2022-10-18 |
| CN115192776B CN115192776B (en) | 2023-08-08 |
Family
ID=83584446
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210860482.2A Active CN115192776B (en) | 2022-07-21 | 2022-07-21 | Method for preparing tough hydrogel for repairing tendon injury |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115192776B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117599236A (en) * | 2024-01-24 | 2024-02-27 | 北京康派特医疗器械有限公司 | Preparation method of absorbable tissue adhesive for blood vessels and organs |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111773431A (en) * | 2020-06-17 | 2020-10-16 | 北京大学口腔医学院 | Preparation method of tendon-like scaffold material and application of tendon-like scaffold material in tendon injury regeneration and repair |
| CN113230449A (en) * | 2021-05-06 | 2021-08-10 | 西北大学 | Glucose and enzyme double-response dressing for treating chronic wound surface of diabetes and preparation method thereof |
| CN113476660A (en) * | 2021-05-17 | 2021-10-08 | 重庆医科大学 | Preparation method of highly-bionic composite scaffold simulating tendon-bone interface |
-
2022
- 2022-07-21 CN CN202210860482.2A patent/CN115192776B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111773431A (en) * | 2020-06-17 | 2020-10-16 | 北京大学口腔医学院 | Preparation method of tendon-like scaffold material and application of tendon-like scaffold material in tendon injury regeneration and repair |
| CN113230449A (en) * | 2021-05-06 | 2021-08-10 | 西北大学 | Glucose and enzyme double-response dressing for treating chronic wound surface of diabetes and preparation method thereof |
| CN113476660A (en) * | 2021-05-17 | 2021-10-08 | 重庆医科大学 | Preparation method of highly-bionic composite scaffold simulating tendon-bone interface |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117599236A (en) * | 2024-01-24 | 2024-02-27 | 北京康派特医疗器械有限公司 | Preparation method of absorbable tissue adhesive for blood vessels and organs |
| CN117599236B (en) * | 2024-01-24 | 2024-04-23 | 北京康派特医疗器械有限公司 | Preparation method of absorbable tissue adhesive for blood vessels and organs |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115192776B (en) | 2023-08-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106310383B (en) | Injectable bone repair hydrogel and preparation method thereof | |
| Yan et al. | Facile preparation of bioactive silk fibroin/hyaluronic acid hydrogels | |
| JP2505312B2 (en) | Collagen-Polymer-conjugate | |
| CN113549227B (en) | Chemical crosslinking hydrogel and microsphere thereof, preparation method and application | |
| CN110743038B (en) | Double-network structure composite hydrogel and preparation method and application thereof | |
| CN109966552B (en) | Tissue engineering scaffold material and preparation method, application and application method thereof | |
| Wang et al. | Enhanced physical and biological properties of chitosan scaffold by silk proteins cross-linking | |
| CN110760103B (en) | Viscoelastic hydrogel and preparation method and application thereof | |
| CN106822183A (en) | A kind of photosensitive platelet rich plasma gel and its production and use | |
| CN1235646C (en) | Method for preparing multifunctional biological repair material | |
| CN110548171B (en) | Gelatin-based bone tissue adhesive, and preparation method and application thereof | |
| CN113999404B (en) | Preparation method of double-crosslinked stem cell sphere hydrogel for osteoarthritis | |
| CN113209363A (en) | Adhesive hemostasis injectable chitosan gel and preparation method and application thereof | |
| CN111407921A (en) | Medical hydrogel dressing, and preparation method and application thereof | |
| US9993525B2 (en) | Biomaterial of chymotryptically isolated fraction of fibroin in a hydrogel | |
| CN112587721A (en) | Injection filling material and preparation process thereof | |
| CN115192776B (en) | Method for preparing tough hydrogel for repairing tendon injury | |
| CN111330075B (en) | Preparation method and application of squid type II gelatin double-network hydrogel | |
| CN113877001A (en) | Silk fibroin composite gel for injection | |
| Wang et al. | 3D printed chondrogenic functionalized PGS bioactive scaffold for cartilage regeneration | |
| CN110448727B (en) | Adhesive hydrogel material, suture-free artificial nerve conduit and preparation method thereof | |
| CN111821513A (en) | Composite hydrogel for promoting cartilage formation and preparation method and application thereof | |
| RU2715715C1 (en) | Sterile transparent concentrated solution of biocompatible collagen, method for production and use thereof | |
| CN112007210A (en) | Photoinitiated polyethylene glycol-based hydrogel dressing and preparation method thereof | |
| US20120003176A1 (en) | Hydrogel-forming composition comprising natural and synthetic segments |
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 |