CN116808279A - Hydrophilic composite collagen sponge and preparation method thereof - Google Patents
Hydrophilic composite collagen sponge and preparation method thereof Download PDFInfo
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Classifications
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- 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
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
- A61L24/10—Polypeptides; Proteins
- A61L24/102—Collagen
-
- 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
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/001—Use of materials characterised by their function or physical properties
- A61L24/0036—Porous materials, e.g. foams or sponges
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- 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
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
- A61L24/08—Polysaccharides
-
- 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
- C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment 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
- C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
- C08J9/36—After-treatment
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L89/00—Compositions of proteins; Compositions of derivatives thereof
-
- 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
- A61L2400/04—Materials for stopping bleeding
-
- 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
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Epidemiology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Surgery (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Materials Engineering (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Dispersion Chemistry (AREA)
- Materials For Medical Uses (AREA)
Abstract
The invention relates to a hydrophilic composite collagen sponge and a preparation method thereof, and the hydrophilic composite collagen sponge specifically comprises the following components: firstly, obtaining collagen stock solution A; the collagen stock solution A is modified to obtain water-soluble collagen stock solution B; drawing pretreatment is carried out on the water-soluble collagen stock solution B, and then freeze drying is carried out to obtain a hydrophilic collagen drawing basal membrane D; uniformly spreading the collagen stock solution A on a collagen wiredrawing base film D, performing wiredrawing pretreatment, and freeze-drying to obtain a collagen wiredrawing sponge E; uniformly spreading the chitosan solution C on the collagen wiredrawing sponge E, and freeze-drying to obtain the hydrophilic composite collagen sponge. The collagen sponge has two characteristic contact surfaces, the modified collagen contact surface has better hydrophilicity, the chitosan contact surface has sterilization and bacteriostasis effects, and the middle collagen sponge layer has the characteristics of triple helix structure, stable biological activity, excellent tensile property, difficult breakage and good clinical application effect.
Description
Technical Field
The invention belongs to the technical field of biomedical materials, and particularly relates to a hydrophilic composite collagen sponge and a preparation method thereof.
Background
Collagen widely exists on the skin, tendons, bones and other parts of organisms, and for a human body, the collagen accounts for 6 percent of the weight of the human body, is the protein with the most abundant content in the human body, accounts for 25 to 30 percent of the protein content in the human body, plays roles in protecting, supporting and the like in the human body, has a unique triple helix structure, has excellent biocompatibility, and is mainly used in the fields of medical materials, cosmetics, food industry and the like.
Collagen is mainly used for wound hemostasis, subcutaneous tissue filling and the like in the field of medical instruments, the main product form of the collagen for intraoperative hemostasis is spongy, and the collagen sponge has good blood sucking and hemostasis performances and promotes tissue repair and healing. The collagen sponge can effectively promote the healing of wounds while playing a good hemostatic property, so that the collagen sponge has a good application prospect in clinical hemostasis.
The collagen molecule contains a large number of active groups such as hydroxyl, carboxyl, amino and the like, so that the active groups on the collagen are modified, and the increase of the hydrophilicity and antithrombotic property of the collagen becomes a hot spot of current research. By introducing hydrophilic modification groups on the side chains of the collagen, the collagen has higher water absorbability, and meanwhile, the negative charge on the surface of the collagen is greatly increased, so that the adhesion of platelets can be reduced, and certain antithrombotic performance is presented.
In the prior art, most of collagen sponge is prepared by compounding collagen and chitosan, the preparation process generally has a trend of complicating, and the related sponge performance also has room for improvement and improvement.
For example, CN107233613B discloses high-purity high-activity medical collagen from aquatic organism sources, which solves the problems of poor mechanical strength, weak degradation resistance and susceptibility of wounds to microbial infection of natural collagen dressing. The method for removing terminal peptide by combining acid extraction with pepsin is adopted to extract collagen, the extraction rate is improved by the method of ultrahigh pressure treatment and tissue trituration, and the high-purity and high-activity medical collagen is prepared by a series of operations of cell removal, impurity protein removal, degreasing, multiple salting-out, dialysis, sterilization, decolorization, heat source removal and the like for purification. The method comprises the steps of preparing a network-shaped collagen sponge with uniform pores through a vacuum freeze drying process, modifying the collagen sponge in a physical crosslinking and chemical crosslinking mode to prepare an inner layer, compositing the inner layer with a chitosan outer layer film and a medical non-woven fabric base cloth layer, and finally sterilizing by Co-60 to obtain a composite multi-layer medical dressing finished product.
For example, CN105327383A relates to a hemostatic biological material-collagen/calcium alginate/chitosan composite hemostatic sponge and a preparation method thereof. The hemostatic sponge is prepared by dissolving collagen, calcium alginate and chitosan according to a certain proportion and adopting a freeze drying method. After the composite hemostatic sponge is contacted with a wound surface, platelets can be quickly aggregated to form a blood crust, and the good liquid absorption property of the composite hemostatic sponge enables the blood crust to absorb a large amount of exudates to generate certain pressure on the wound, so that a certain compression hemostasis effect is achieved, and a large amount of Ca is contained in the blood crust 2+ Can be contacted with the wound surface to quickly contact with Na in blood + Exchange occurs, so that the generation of thrombin is promoted, and the self-coagulation of the wound surface is accelerated.
As in US20180169293A1, relates to a hydrolyzed collagen dough produced from bovine collagen and a method for producing a collagen dough from bovine hide. The collagen dough comprises water, bovine collagen, sodium benzoate, and hydrochloric acid. To produce collagen dough, the skin layers are separated from the cowhide and treated. Collagen dough can be used in biomedical applications such as cell technology/tissue engineering, suture materials or films and sponges for skin grafts. The collagen dough may further comprise chitosan.
Based on the above, a preparation method of a hydrophilic composite collagen sponge is needed, the preparation process is relatively simple, and meanwhile, the product has excellent performance, better hydrophilicity, high tensile strength and excellent antibacterial and bacteriostatic properties.
Disclosure of Invention
The invention aims to provide a brand-new preparation process of a hydrophilic composite collagen sponge, which is simple to operate in industrial production and can be used for producing the collagen sponge with good tensile property, hydrophilicity, hemostasis property and antibacterial property.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the invention provides a preparation method of a hydrophilic composite collagen sponge, which comprises the following steps:
s1: pretreating animal tissues, inactivating viruses, extracting collagen by using an acid enzyme, and salting out and purifying to obtain a collagen stock solution A;
s2: the collagen stock solution A is subjected to hydrophilic grafting modification, modified collagen precipitation, cleaning and redissolution to obtain water-soluble collagen stock solution B;
s3: preparing chitosan to obtain chitosan solution C;
s4: uniformly spreading the water-soluble collagen stock solution B in a freeze dryer die, performing wire drawing pretreatment, and then performing freeze drying to prepare a hydrophilic collagen wire drawing base film D;
s5: uniformly spreading the collagen stock solution A onto the hydrophilic collagen wiredrawing base film D, performing wiredrawing pretreatment, and then performing freeze drying to obtain a collagen wiredrawing sponge E;
s6: uniformly spreading the chitosan solution C on the collagen wiredrawing sponge E, and freeze-drying to obtain the hydrophilic composite collagen sponge.
As a further improvement of the present invention, the animal tissue in S1 is bovine achilles tendon, and the pretreatment in S1 includes slicing treatment after removing superficial fat.
As a further improvement of the invention, the virus inactivation treatment in the step S1 is carried out by soaking the pretreated material for 0.5-2h by using 0.5-1M sodium hydroxide, and washing the pretreated material with purified water for multiple times; the step S1 of extracting collagen by the acid enzyme is specifically carried out according to animal tissues: the mass ratio of pepsin is (10-30) 1, extracting with acid enzyme; the salting-out and purifying method in the step S1 is sodium chloride dialysis, the adding concentration of sodium chloride is 40-50 g/L, and the dialysis time is 12-36h.
As a further improvement of the invention, the modifier adopted in the hydrophilic grafting modification process in the step S2 is succinic anhydride, and the solvent is dimethyl sulfoxide.
As a further improvement of the invention, the isoelectric point of the precipitation of the modified collagen in the S2 is pH=4.1-4.9, and the concentration of the water-soluble collagen stock solution B in the S2 is 5-20mg/mL.
As a further improvement of the invention, the concentration of the chitosan solution C in the S3 is 1-10mg/mL.
As a further improvement of the invention, the thickness of the water-soluble collagen stock solution B paved in the S4 into the mold of the freeze dryer is 1-5mm, and the wire drawing pretreatment in the S4 is as follows: and (3) carrying out negative pressure and normal pressure repeated wiredrawing treatment on the water-soluble collagen stock solution B by utilizing negative pressure vacuum of a freeze dryer, wherein the specific parameters are as follows: the vacuum degree of the negative pressure is-60 Pa to-100 Pa; the holding time of the negative pressure is 20-50min, the holding time of the normal pressure is 20-50min, and the repetition times of the negative pressure and the normal pressure are 1-5 times; the core of the wire drawing pretreatment process is that after negative pressure vacuumizing, the wire drawing pretreatment process instantaneously becomes normal pressure, and wire drawing can occur on the surface of the water-soluble collagen stock solution B;
the freeze drying in the step S4 comprises prefreezing, sublimation drying and analysis drying, and the specific process parameters are as follows:
the pre-freezing temperature is minus 35 ℃ to minus 45 ℃, the cooling rate is 1-2 ℃/min, and the pre-freezing time is 0.5-2h; the temperature of sublimation drying is-10 to-25 ℃, and the period of the sublimation drying is 5-10 hours; the vacuum degree of negative pressure in the sublimation drying is minus 25 Pa to minus 45Pa; the temperature of the analytic drying is 30-40 ℃, and the time of the analytic drying is 1-3h; the total time of freeze drying is 7-18h.
As a further improvement of the invention, the thickness of the collagen stock solution A paved on the hydrophilic collagen wiredrawing base film D in the step S5 is 5-10mm; the wire drawing pretreatment in the step S5 is as follows: and (3) carrying out negative pressure and normal pressure repeated wiredrawing treatment on the collagen stock solution A by utilizing negative pressure vacuum of a freeze dryer, wherein the specific parameters are as follows: the vacuum degree of the negative pressure is-70 Pa to-100 Pa; the holding time of the negative pressure is 30-120min, the holding time of the normal pressure is 20-50min, and the repetition times of the negative pressure and the normal pressure are 1-5 times; the core of the wire drawing pretreatment process is that after negative pressure vacuumizing, the surface of the collagen stock solution A is instantaneously changed into normal pressure, and wire drawing can occur;
the freeze drying in the step S5 comprises prefreezing, sublimation drying and analysis drying, and the specific process parameters are as follows:
the pre-freezing temperature is minus 35 ℃ to minus 45 ℃, the cooling rate of the pre-freezing is 1-2 ℃/min, and the pre-freezing time is 2-4h; the temperature of sublimation drying in the step S5 is-10 to-25 ℃, and the period of sublimation drying is 10-20 hours; the negative pressure vacuum degree in the sublimation drying is-25 to-45 Pa; the temperature of the desorption drying is 30-40 ℃, and the duration of the desorption drying is 1-5h; the total time of freeze drying is 36-72h;
the concentration of the collagen stock solution A in the step S5 is 10-25mg/mL.
As a further improvement of the invention, the thickness of the chitosan solution C paved on the collagen wiredrawing sponge E in the step S6 is 1-5mm;
the freeze drying in the step S6 comprises prefreezing, sublimation drying and analysis drying, and the specific process parameters are as follows:
the pre-freezing temperature in the step S6 is minus 35 ℃ to minus 45 ℃, the cooling rate of the pre-freezing is 1-2 ℃/min, and the pre-freezing time is 0.5-2h; the temperature of sublimation drying in the step S6 is-10 to-25 ℃, and the period of sublimation drying is 5-10 hours; the vacuum degree of negative pressure in the sublimation drying is minus 25 Pa to minus 45Pa; the temperature of the desorption drying is 30-40 ℃, and the duration of the desorption drying is 0.5-2h; the total time of freeze drying is 6-18h.
The invention provides a hydrophilic composite collagen sponge prepared by the preparation method of the hydrophilic composite collagen sponge.
The invention provides a preparation method of a hydrophilic composite collagen sponge, which is characterized by researching modification technology of a collagen stock solution B, concentration searching of the collagen stock solution A, searching of freeze-drying parameters of the collagen stock solution and a chitosan solution, freeze-drying thickness and the like; meanwhile, the inventor unexpectedly discovers that the sponge prepared by freeze-drying after the base film and the collagen stock solution are mixed can have stronger bonding capability between the base film and the sponge, so that the physical property of the composite collagen sponge is better, and adverse events such as chip and slag falling in the clinical use process are avoided. Meanwhile, the chitosan surface layer is compounded, so that the antibacterial and bacteriostatic ability of the sponge is improved. The invention can prepare the composite collagen sponge with better hydrophilicity, high tensile strength and excellent antibacterial and bacteriostatic properties. The beneficial effects of adopting above-mentioned technical scheme to produce lie in:
the hydrophilic composite collagen sponge prepared by the preparation method provided by the invention has stronger hydrophilicity and excellent hemostatic effect in clinical use.
According to the invention, a novel preparation process of the hydrophilic collagen sponge is established by exploring a collagen extraction and modification process, and the prepared hydrophilic composite collagen sponge has better hydrophilicity.
According to the process, the hydrophilic collagen stock solution is subjected to negative pressure vacuum wire drawing treatment by using a freeze dryer, any adhesive is not used, the adhesive force between the base film and the sponge is increased by using the characteristics of substances, the prepared composite collagen sponge has strong tensile property, and adverse events such as chip and slag falling in the clinical use process are avoided.
The chitosan film layer is added in the process, so that the antibacterial and bacteriostatic ability of the collagen sponge is improved, and the process has more advantages in clinical use.
Drawings
FIG. 1 is a specific process flow diagram of example 1;
FIG. 2 is a photograph of a hydrophilic composite collagen sponge obtained in example 1;
FIG. 3 is a photograph of a hydrophilic collagen drawing base film D obtained in example 1;
FIG. 4 is a photograph of a collagen drawing sponge E obtained in example 1;
FIG. 5 is an infrared spectrum of the hydrophilic composite collagen sponge obtained in example 1;
FIG. 6 is an SDS-PAGE gel of the hydrophilic composite collagen sponge obtained in example 1.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be clearly and completely described in connection with the following specific embodiments.
Unless otherwise indicated, the equipment used in this embodiment is conventional, the reagents used are conventional, all commercially available, and the methods of operation used are described in textbooks in the field.
Example 1 preparation of hydrophilic composite collagen sponge
The preparation process flow chart of the hydrophilic composite collagen sponge is shown in figure 1, and the specific process is as follows:
s1: pretreating animal tissues, inactivating viruses, extracting collagen by using an acid enzyme, and salting out and purifying to obtain a collagen stock solution A;
pretreatment of bovine Achilles tendon: the animal tissue bovine Achilles tendon is washed, and then the surface layer grease is removed, and the slicing treatment is carried out.
Virus inactivation treatment: the tissue after slicing was immersed in 0.5M sodium hydroxide solution, and after 2 hours, the tissue slices were repeatedly rinsed with purified water, and the slices were further minced into a paste.
Extracting collagen by an acidosis method: the minced tissue is weighed and then put into a reaction kettle, pepsin (the added amount is 1/20 of the weight of the tissue) and acetic acid (the added amount is 2.5% of the weight of the tissue) are added, and the crude collagen solution is obtained after the reaction is carried out for 48 hours at a rotating speed of 160 rpm;
salting out, dialyzing and purifying: adding sodium chloride solution into the crude collagen solution to make the final concentration of the crude collagen solution in the glue solution be 42g/L, separating out collagen in a floccule form at the moment, and centrifuging to discard supernatant; adding 0.3% acetic acid water solution into the floccule to dissolve collagen, placing into a dialysis bag, sealing, placing into purified water, and dialyzing for 24 hr; obtaining purified collagen stock solution A, and determining that the concentration of the collagen stock solution A is 20mg/mL and the purity is 96.5%;
s2: the collagen stock solution A is subjected to hydrophilic grafting modification, modified collagen precipitation, cleaning and redissolution to obtain water-soluble collagen stock solution B;
hydrophilic modification: taking a proper amount of collagen stock solution A, diluting with 0.3% acetic acid to obtain a collagen solution with the concentration of 10mg/mL, adjusting the pH value to 12, adding a modifier succinic anhydride, wherein the mass ratio of the succinic anhydride to the collagen is 1:1, and stirring for 3 hours to obtain a modified collagen crude solution;
precipitation of modified collagen: adding 5M sodium hydroxide solution into the modified collagen crude solution to enable the pH value to be 4.5, and separating out water-soluble collagen solid;
cleaning and redissolving: repeatedly cleaning the water-soluble collagen solid with 5% sodium chloride solution for 3 times, centrifuging, discarding supernatant, and dissolving with 0.3% acetic acid to obtain water-soluble collagen stock solution B. The concentration of the water-soluble collagen stock solution B is measured to be 10mg/mL, and the purity is measured to be 97.6%.
S3: preparing a chitosan solution C;
chitosan solution C: the chitosan concentration in chitosan solution C was 5mg/mL.
S4: uniformly spreading the water-soluble collagen stock solution B in a freeze dryer die, performing wire drawing pretreatment, and then performing freeze drying to prepare a hydrophilic collagen wire drawing base film D;
hydrophilic collagen wiredrawing basement membrane D: the water-soluble collagen stock solution B with the concentration of 10mg/mL is put into a freeze dryer die, and the paving thickness is 3mm; the water-soluble collagen stock solution B is subjected to negative pressure and normal pressure repeated wiredrawing treatment by utilizing the negative pressure vacuum of a freeze dryer, and the specific parameters are as follows: the vacuum degree of the negative pressure is-80 Pa; the negative pressure holding time is 30min, the normal pressure holding time is 30min, and the negative pressure and normal pressure repetition times are 3 times. The core of the wire drawing pretreatment process is that after the vacuum pumping is carried out under the negative pressure, the pressure is changed into normal pressure instantly, and the wire drawing can occur on the surface of the water-soluble collagen stock solution B.
The pre-freezing temperature is-40 ℃, the cooling rate of the pre-freezing is 1 ℃/min, and the freezing time is 1h; the sublimation drying temperature is-20 ℃, and the duration of the sublimation drying stage is 8 hours; the vacuum degree of negative pressure is-40 Pa during sublimation drying; the analytic drying temperature is 35 ℃, and the analytic drying time is 1h; the total time of freeze drying is 12 hours, and the hydrophilic collagen wiredrawing base film D is obtained, and the photo of the prepared hydrophilic collagen wiredrawing base film D is shown in figure 3.
S5: uniformly spreading the collagen stock solution A on a hydrophilic collagen wiredrawing base film D, performing wiredrawing pretreatment, and then freeze-drying to obtain a collagen wiredrawing sponge E, wherein a photo of the prepared collagen wiredrawing sponge E is shown in fig. 4;
collagen wiredrawing sponge E: spraying the collagen stock solution A with the concentration of 20mg/mL onto the hydrophilic collagen drawing base film D, wherein the thickness of the collagen stock solution A is 8mm; the freeze-drying wiredrawing is carried out with negative pressure and normal pressure repeatedly wiredrawing treatment, and the specific parameters are as follows: the vacuum degree of the negative pressure is-80 Pa, the holding time of the negative pressure is 60min, the holding time of the normal pressure is 30min, and the treatment is repeated for 3 times;
the pre-freezing temperature is-40 ℃, the cooling rate of the pre-freezing is 1 ℃/min, and the pre-freezing time is 3 hours; the sublimation drying temperature is-20 ℃, and the duration of the sublimation drying stage is 16 hours; the vacuum degree of negative pressure is-40 Pa during sublimation drying; the analytic drying temperature is 35 ℃, and the analytic drying time is 3 hours; and (5) freeze drying for 48 hours to obtain the collagen wiredrawing sponge E.
S6: uniformly spreading the chitosan solution C on the collagen wiredrawing sponge E, and freeze-drying to obtain the hydrophilic composite collagen sponge.
Hydrophilic composite collagen sponge: the chitosan solution C is uniformly sprayed on the collagen wiredrawing sponge E, and the freeze drying parameters are as follows:
the pre-freezing temperature is-45 ℃, the cooling rate of the pre-freezing is 2 ℃/min, and the pre-freezing time is 1h; the sublimation drying temperature is-20 ℃, and the duration of the sublimation drying stage is 8 hours; the vacuum degree of negative pressure is-45 Pa during sublimation drying; the analytic drying temperature is 30 ℃, and the analytic drying time is 1h; the total time of freeze drying is 12 hours, and the hydrophilic composite collagen sponge is obtained, and the photo of the prepared hydrophilic composite collagen sponge is shown in figure 2.
Example 2 preparation of hydrophilic composite collagen sponge
The only difference compared to example 1 is that the solution concentration of chitosan solution C is 1mg/mL.
Example 3 preparation of hydrophilic composite collagen sponge
The only difference compared to example 1 is that the solution concentration of chitosan solution C is 3mg/mL.
EXAMPLE 4 preparation of hydrophilic composite collagen sponge
The only difference compared to example 1 is that the solution concentration of chitosan solution C was 8mg/mL.
Example 5 preparation of hydrophilic composite collagen sponge
The only difference compared to example 1 is that the solution concentration of chitosan solution C is 10mg/mL.
EXAMPLE 6 preparation of hydrophilic composite collagen sponge
The difference from example 1 is only that the isoelectric point of collagen precipitation after modification in S2 is ph=4.1.
EXAMPLE 7 preparation of hydrophilic composite collagen sponge
The difference from example 1 is only that the isoelectric point of collagen precipitation after modification in S2 is ph=4.3.
Example 8 preparation of hydrophilic composite collagen sponge
The difference from example 1 is only that the isoelectric point of collagen precipitation after modification in S2 is ph=4.7.
Example 9 preparation of hydrophilic composite collagen sponge
The difference from example 1 is only that the isoelectric point of collagen precipitation after modification in S2 is ph=4.9.
EXAMPLE 10 preparation of hydrophilic composite collagen sponge
The difference compared with example 1 is only that the wiredrawing lyophilization parameters in S4 are different, specifically that the concentration of the water-soluble collagen dope B is 5mg/mL;
EXAMPLE 11 preparation of hydrophilic composite collagen sponge
The difference compared with example 1 is only that the wiredrawing lyophilization parameters in S4 are different, specifically that the concentration of the water-soluble collagen dope B is 8mg/mL;
EXAMPLE 12 preparation of hydrophilic composite collagen sponge
The difference compared with example 1 is only that the wiredrawing lyophilization parameters in S4 are different, specifically that the concentration of the water-soluble collagen dope B is 20mg/mL;
EXAMPLE 13 preparation of hydrophilic composite collagen sponge
Compared with example 1, the difference is only that the wiredrawing freeze-drying parameters in S4 are different, in particular, the tiling thickness is 1mm;
EXAMPLE 14 preparation of hydrophilic composite collagen sponge
Compared with example 1, the difference is only that the wiredrawing freeze-drying parameters in S4 are different, in particular, the tiling thickness is 4mm;
EXAMPLE 15 preparation of hydrophilic composite collagen sponge
Compared with example 1, the difference is only that the wiredrawing freeze-drying parameters in S4 are different, in particular, the tiling thickness is 5mm;
EXAMPLE 16 preparation of hydrophilic composite collagen sponge
Compared with the embodiment 1, the difference is that the drawing freeze-drying parameters in S4 are different, and specifically, the negative pressure vacuum degree of the drawing process is-60 Pa;
EXAMPLE 17 preparation of hydrophilic composite collagen sponge
Compared with the embodiment 1, the difference is that the drawing freeze-drying parameters in S4 are different, and specifically, the negative pressure vacuum degree of the drawing process is-90 Pa;
EXAMPLE 18 preparation of hydrophilic composite collagen sponge
Compared with the embodiment 1, the difference is that the drawing freeze-drying parameters in S4 are different, and specifically, the negative pressure vacuum degree of the drawing process is-100 Pa;
EXAMPLE 19 preparation of hydrophilic composite collagen sponge
Compared with example 1, the difference is only that the wiredrawing freeze-drying parameters in S4 are different, and specifically, the negative pressure holding time is 20min;
EXAMPLE 20 preparation of hydrophilic composite collagen sponge
Compared with example 1, the difference is only that the wiredrawing freeze-drying parameters in S4 are different, and specifically, the negative pressure holding time is 40min;
EXAMPLE 21 preparation of hydrophilic composite collagen sponge
Compared with example 1, the difference is only that the wiredrawing freeze-drying parameters in S4 are different, and specifically, the negative pressure holding time is 50min;
EXAMPLE 22 preparation of hydrophilic composite collagen sponge
Compared with example 1, the difference is only that the wiredrawing freeze-drying parameters in S4 are different, and in particular, the normal pressure holding time is 20min;
EXAMPLE 23 preparation of hydrophilic composite collagen sponge
Compared with example 1, the difference is only that the wiredrawing freeze-drying parameters in S4 are different, and in particular, the normal pressure holding time is 40min;
EXAMPLE 24 preparation of hydrophilic composite collagen sponge
Compared with example 1, the difference is only that the wiredrawing freeze-drying parameters in S4 are different, and in particular, the normal pressure holding time is 50min;
EXAMPLE 25 preparation of hydrophilic composite collagen sponge
The difference compared to example 1 is only that the wire-drawing lyophilization parameters in S4 are different, specifically, the number of treatments is 1;
EXAMPLE 26 preparation of hydrophilic composite collagen sponge
The difference compared to example 1 is only that the wire-drawing lyophilization parameters in S4 are different, specifically, the number of treatments is 5;
EXAMPLE 27 preparation of hydrophilic composite collagen sponge
The difference compared with example 1 is only that the wiredrawing lyophilization parameters in S5 are different, specifically that the collagen stock solution a concentration is 10mg/mL;
EXAMPLE 28 preparation of hydrophilic composite collagen sponge
The difference compared with example 1 is only that the wiredrawing lyophilization parameters in S5 are different, specifically that the collagen stock solution a concentration is 15mg/mL;
EXAMPLE 29 preparation of hydrophilic composite collagen sponge
The difference compared with example 1 is only that the wiredrawing lyophilization parameters in S5 are different, specifically that the collagen stock solution a concentration is 25mg/mL;
EXAMPLE 30 preparation of hydrophilic composite collagen sponge
Compared with example 1, the difference is only that the wiredrawing freeze-drying parameters in S5 are different, in particular, the tiling thickness is 7mm;
EXAMPLE 31 preparation of hydrophilic composite collagen sponge
Compared with example 1, the difference is only that the wiredrawing freeze-drying parameters in S5 are different, in particular, the tiling thickness is 9mm;
EXAMPLE 32 preparation of hydrophilic composite collagen sponge
Compared with example 1, the difference is only that the wiredrawing freeze-drying parameters in S5 are different, in particular, the tiling thickness is 10mm;
EXAMPLE 33 preparation of hydrophilic composite collagen sponge
Compared with the embodiment 1, the difference is that the drawing freeze-drying parameters in S5 are different, and in particular, the negative pressure vacuum degree of the drawing process is-70 Pa;
EXAMPLE 34 preparation of hydrophilic composite collagen sponge
Compared with the embodiment 1, the difference is that the drawing freeze-drying parameters in S5 are different, and in particular, the negative pressure vacuum degree of the drawing process is-90 Pa;
example 35 preparation of hydrophilic composite collagen sponge
Compared with the embodiment 1, the difference is that the drawing freeze-drying parameters in S5 are different, and specifically, the negative pressure vacuum degree of the drawing process is-100 Pa;
EXAMPLE 36 preparation of hydrophilic composite collagen sponge
Compared with example 1, the difference is only that the wiredrawing freeze-drying parameters in S5 are different, specifically that the negative pressure holding time is 30min;
EXAMPLE 37 preparation of hydrophilic composite collagen sponge
Compared with example 1, the difference is only that the wiredrawing freeze-drying parameters in S5 are different, and specifically, the negative pressure holding time is 90min;
EXAMPLE 38 preparation of hydrophilic composite collagen sponge
Compared with example 1, the difference is only that the wiredrawing freeze-drying parameters in S5 are different, and specifically, the negative pressure holding time is 120min;
EXAMPLE 39 preparation of hydrophilic composite collagen sponge
Compared with example 1, the difference is only that the wiredrawing freeze-drying parameters in S5 are different, and in particular, the normal pressure holding time is 20min;
EXAMPLE 40 preparation of hydrophilic composite collagen sponge
Compared with example 1, the difference is only that the wiredrawing freeze-drying parameters in S5 are different, and in particular, the normal pressure holding time is 40min;
EXAMPLE 41 preparation of hydrophilic composite collagen sponge
Compared with example 1, the difference is only that the wiredrawing freeze-drying parameters in S5 are different, and in particular, the normal pressure holding time is 50min;
EXAMPLE 42 preparation of hydrophilic composite collagen sponge
The difference compared to example 1 is only that the wire drawing lyophilization parameters in S5 are different, specifically, the number of treatments is 1;
EXAMPLE 43 preparation of hydrophilic composite collagen sponge
The difference compared with example 1 is only that the wire drawing lyophilization parameters in S5 are different, specifically, the number of repeated treatments is 5 times;
comparative example 1
The test of comparative example 1 was identical to the test conditions of example 1 except that comparative example 1 did not contain the preparation of step S5 collagen drawing sponge E, and chitosan solution C was directly sprayed uniformly on hydrophilic collagen drawing base film D.
Comparative example 2
The test described in comparative example 2 was identical to the test conditions described in example 1, except that comparative example 2 did not contain step S6.
Comparative example 3:
the test described in comparative example 3 was identical to the test conditions described in comparative document 1, except that comparative example 3 did not contain step S2, i.e., the starting material of the base film D was also collagen dope a.
Comparative example 4
Compared with the embodiment 1, the wiredrawing freeze-drying parameters in the S4 are different, specifically, the wiredrawing process is not adopted, and the wiredrawing freeze-drying parameters are directly pre-frozen;
comparative example 5
Compared with the embodiment 1, the wiredrawing freeze-drying parameters in S5 are different, specifically, the wiredrawing process is not adopted, and the wiredrawing freeze-drying parameters are directly pre-frozen;
test 1 hydrophilic composite collagen sponge finished product appearance observation
See table 1 for specific results by observing the procedure and the products of the examples and the comparative examples.
Test 2 liquid absorbing Performance test of hydrophilic composite collagen sponge finished product
The collagen sponges obtained in examples and comparative examples were subjected to liquid absorption performance test as follows:
taking square with the size of 2cm of collagen sponge, measuring the weight of the square, marking the square as m1, putting the square into water, slightly wetting the square with fingers to be completely wetted, slightly holding forceps and draining the square on the water surface for 1min, weighing the square as m2, and measuring the water absorption performance= (m 2-m 1)/m 1 by the same method for 10 times, and taking the average value of the square to obtain the water absorption performance. See table 1 for specific results.
Test 3 collagen sponge tensile Property test
The collagen sponges obtained in examples and comparative examples were subjected to tensile property test, the sponges were made into a bar shape 1cm wide and 10cm long, both ends were clamped with a jig, a tensile force was applied thereto until breaking, and a tensile force value at breaking was recorded. See table 1 for test results:
table 1 test data for the properties of collagen sponges obtained in examples and comparative examples
Test 4 hemostatic Performance test of collagen sponge finished product
The hydrophilic composite collagen sponges obtained in examples and comparative examples were subjected to hemostatic performance test as follows:
a miniature pig is used as an animal model; after the skin preparation of the experimental animal, the femoral artery/jugular vein is exposed, an incision with the length of 1cm is made, and after the incision is sutured, the experimental animal is free to bleed for 2 seconds, thus completing the model establishment. Then covering the bleeding points with collagen sponge, and recording the hemostatic time and the bleeding amount;
since the hemostatic performance of the collagen sponge is evaluated by adopting an animal model in the test example, in view of the protection principle of the test animal, the hemostatic test investigation is carried out by selecting the test samples obtained by the examples and the comparative examples related to the hemostatic performance of the product, and specific data are shown in the following table 2:
table 2 collagen sponge hemostatic performance test data
Test 5 quality evaluation of hydrophilic composite collagen sponge
In the technical process of preparing the hydrophilic composite collagen sponge by the preparation process, the collagen stock solutions A and B are type I collagen, and in order to characterize the characteristics of the product prepared by the preparation process, the quality evaluation items of the collagen sponge listed in the test example comprise infrared spectrum identification, SDS-PAGE gel electrophoresis identification and type I collagen purity detection.
The infrared spectrum identification detection method refers to 0402 infrared spectrophotometry in four parts of Chinese pharmacopoeia, a collagen sponge sample prepared in the embodiment 1 is taken and added into potassium bromide powder for grinding and tabletting, a sample to be detected is detected on a machine after background scanning is carried out by using the potassium bromide tablet, the detected infrared spectrum is shown in figure 5 of the attached drawing of the specification, and the collagen sponge of the sample to be detected has the following infrared spectrum characteristic peaks: 3488-3188cm -1 、3102-3062cm -1 、2985-2915cm -1 、1667-1627 cm -1 、1548 cm -1 、1452 cm -1 、1402 cm -1 、1338 cm -1 、1238 cm -1 The method comprises the steps of carrying out a first treatment on the surface of the Meets the infrared spectrum characteristics of the type I collagen.
The SDS-PAGE gel electrophoresis identification detection method is carried out by referring to the method specified by the fourth method of the 0541 electrophoresis method in the fourth section of Chinese pharmacopoeia, the concentration of the separating gel is 7% by SDS-PAGE electrophoresis, the sample adding amount is not less than 20 mug, the sponge of the example is taken as a sample, and the sample and a reference substance (marked reference substance) are analyzed by SDS-PAGE to obtain electrophoresis bands, and the result is shown in figure 6.
FIG. 6 shows the electrophoresis bands obtained by performing the two parallel test conditions of example 1, wherein the alpha 1 and alpha 2 bands are obvious, and are typical type I collagen profile characteristics, which indicate that the collagen obtained in the process of the invention is type I collagen with a bioactive triple helix structure.
The purity detection method of the type I collagen refers to a method in an annex A of a characterization method of the type I collagen of a medical appliance product of GB/T1453-2016 tissue engineering, SDS-PAGE is used for detecting the purity of the type I collagen, and the detection principle is that after a specific triple helix structure in the collagen is acted by specific collagenase, the total amount of the impurity proteins contained in a collagen sample is detected through determining the dyeing limit of coomassie brilliant blue on Bovine Serum Albumin (BSA).
The specific detection steps are as follows: after preparation of required reagents, electrophoresis and sample loading are carried out, a sample and MaKer are respectively added into sample adding holes, 110V constant-pressure electrophoresis is carried out until strip separation is completed, coomassie brilliant blue reagent is added for dyeing, decoloring treatment is carried out after dyeing for 1h, decolored SDS-PAGE gel is placed on a white board of a developing system, automatic exposure is carried out according to a VISIBLE program, a strip cleaning film is obtained, the optical density of a (greater than 50 kDa) region on a sample lane is calculated by Image J software, and the purity of collagen is calculated, wherein the results are shown in the following table:
TABLE 3 statistics of purity test results for collagen type I
As can be seen from the data in the table, the purity of the collagen stock solution A and the purity of the collagen stock solution B obtained in the embodiment 1 of the invention are both above 95%, and the clinical requirements can be met.
4. I type collagen content detection
The content detection method refers to a third method of a biuret method for a 0731 protein content determination method in four parts of Chinese pharmacopoeia, and the detection principle is as follows: according to the method, more than two peptide bonds contained in protein molecules form a mauve complex with Cu < 2+ > in an alkaline solution, the color depth of the mauve complex is in direct proportion to the protein concentration within a certain range, a protein reference substance solution is used as a standard curve, and the content of protein in a sample is measured by adopting a colorimetric method.
The specific detection steps are as follows: the sample of the example was precisely weighed about 0.5g in a 10mL tube and added with water until 1mL dissolved. Preparing a BSA standard curve, detecting absorbance of a sample and the standard curve at 540nm, bringing the absorbance obtained by the sample into the standard curve, and calculating the collagen content, wherein the result is shown in the following table 4:
TABLE 4 statistics of results of type I collagen content measurements
The quality study of infrared spectrum identification, SDS-PAGE gel electrophoresis identification, purity detection and content detection of the type I collagen is the process detection control of the important raw materials of the collagen A liquid and the collagen B liquid, and aims to ensure that the collagen obtained in the process has a triple helix active structure and the purity meets the product quality requirement.
Although the invention has been described in detail with reference to the foregoing embodiments, those skilled in the art may modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some technical features thereof; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. The preparation method of the hydrophilic composite collagen sponge is characterized by comprising the following steps of:
s1: pretreating animal tissues, inactivating viruses, extracting collagen by using an acid enzyme, and salting out and purifying to obtain a collagen stock solution A;
s2: the collagen stock solution A is subjected to hydrophilic grafting modification, modified collagen precipitation, cleaning and redissolution to obtain water-soluble collagen stock solution B;
s3: preparing chitosan to obtain chitosan solution C;
s4: uniformly spreading the water-soluble collagen stock solution B in a freeze dryer die, performing wire drawing pretreatment, and then performing freeze drying to prepare a hydrophilic collagen wire drawing base film D;
s5: uniformly spreading the collagen stock solution A onto the hydrophilic collagen wiredrawing base film D, performing wiredrawing pretreatment, and then performing freeze drying to obtain a collagen wiredrawing sponge E;
s6: uniformly spreading the chitosan solution C on the collagen wiredrawing sponge E, and freeze-drying to obtain the hydrophilic composite collagen sponge.
2. The method for preparing a hydrophilic composite collagen sponge according to claim 1, wherein the animal tissue in S1 is bovine achilles tendon, and the pretreatment in S1 comprises slicing treatment after removing superficial oil.
3. The method for preparing a hydrophilic composite collagen sponge according to claim 1 or 2, wherein the virus inactivation treatment in S1 is performed by immersing the pretreated material in 0.5-1M sodium hydroxide for 0.5-2 hours, and washing the material with purified water for a plurality of times; the step S1 of extracting collagen by the acid enzyme is specifically carried out according to animal tissues: the mass ratio of pepsin is (10-30) 1, extracting with acid enzyme; the salting-out and purifying method in the step S1 is sodium chloride dialysis, the adding concentration of sodium chloride is 40-50 g/L, and the dialysis time is 12-36h.
4. The method for preparing a hydrophilic composite collagen sponge according to claim 1, wherein the modifying agent adopted in the hydrophilic grafting modification process in S2 is succinic anhydride, and the solvent is dimethyl sulfoxide.
5. The method for preparing a hydrophilic composite collagen sponge according to claim 1 or 4, wherein the isoelectric point of the modified collagen precipitation in S2 is ph=4.1-4.9, and the concentration of the water-soluble collagen stock solution B in S2 is 5-20mg/mL.
6. The method for preparing a hydrophilic composite collagen sponge according to claim 1, wherein the concentration of the chitosan solution C in S3 is 1-10mg/mL.
7. The method for preparing a hydrophilic composite collagen sponge according to claim 1, wherein the thickness of the water-soluble collagen stock solution B in S4 laid in the mold of the freeze dryer is 1-5mm, and the wire drawing pretreatment in S4 is as follows: and (3) carrying out negative pressure and normal pressure repeated wiredrawing treatment on the water-soluble collagen stock solution B by utilizing negative pressure vacuum of a freeze dryer, wherein the specific parameters are as follows: the vacuum degree of the negative pressure is-60 Pa to-100 Pa; the holding time of the negative pressure is 20-50min, the holding time of the normal pressure is 20-50min, and the repetition times of the negative pressure and the normal pressure are 1-5 times; the core of the wire drawing pretreatment process is that after negative pressure vacuumizing, the wire drawing pretreatment process instantaneously becomes normal pressure, and wire drawing can occur on the surface of the water-soluble collagen stock solution B;
the freeze drying in the step S4 comprises prefreezing, sublimation drying and analysis drying, and the specific process parameters are as follows:
the pre-freezing temperature is minus 35 ℃ to minus 45 ℃, the pre-freezing cooling rate is 1-2 ℃/min, and the pre-freezing time is 0.5-2h; the temperature of sublimation drying is-10 to-25 ℃, and the period of the sublimation drying is 5-10 hours; the vacuum degree of negative pressure in the sublimation drying is minus 25 Pa to minus 45Pa; the temperature of the analytic drying is 30-40 ℃, and the time of the analytic drying is 1-3h; the total time of freeze drying is 7-18h.
8. The method for preparing a hydrophilic composite collagen sponge according to claim 1, wherein the thickness of the collagen stock solution a in S5 laid on the hydrophilic collagen drawing base film D is 5-10mm; the wire drawing pretreatment in the step S5 is as follows: and (3) carrying out negative pressure and normal pressure repeated wiredrawing treatment on the collagen stock solution A by utilizing negative pressure vacuum of a freeze dryer, wherein the specific parameters are as follows: the vacuum degree of the negative pressure is-70 Pa to-100 Pa; the holding time of the negative pressure is 30-120min, the holding time of the normal pressure is 20-50min, and the repetition times of the negative pressure and the normal pressure are 1-5 times; the core of the wire drawing pretreatment is that after negative pressure vacuumizing, the surface of the collagen stock solution A is instantaneously changed into normal pressure, and wire drawing can occur;
the freeze drying in the step S5 comprises prefreezing, sublimation drying and analysis drying, and the specific process parameters are as follows:
the pre-freezing temperature is minus 35 ℃ to minus 45 ℃, the cooling rate of the pre-freezing is 1-2 ℃/min, and the pre-freezing time is 2-4h; the temperature of sublimation drying in the step S5 is-10 to-25 ℃, and the period of sublimation drying is 10-20 hours; the negative pressure vacuum degree in the sublimation drying is-25 to-45 Pa; the temperature of the desorption drying is 30-40 ℃, and the duration of the desorption drying is 1-5h; the total time of freeze drying is 36-72h;
the concentration of the collagen stock solution A in the step S5 is 10-25mg/mL.
9. The method for preparing a hydrophilic composite collagen sponge according to claim 1, wherein the thickness of the chitosan solution C in S6 spread on the collagen drawing sponge E is 1-5mm;
the freeze drying in the step S6 comprises prefreezing, sublimation drying and analysis drying, and the specific process parameters are as follows:
the pre-freezing temperature in the step S6 is minus 35 ℃ to minus 45 ℃, the cooling rate of the pre-freezing is 1-2 ℃/min, and the pre-freezing time is 0.5-2h; the temperature of sublimation drying in the step S6 is-10 to-25 ℃, and the period of sublimation drying is 5-10 hours; the vacuum degree of negative pressure in the sublimation drying is minus 25 Pa to minus 45Pa; the temperature of the desorption drying is 30-40 ℃, and the duration of the desorption drying is 0.5-2h; the total time of freeze drying is 6-18h.
10. A hydrophilic composite collagen sponge prepared by the method for preparing a hydrophilic composite collagen sponge according to any one of claims 1 to 9.
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