Preparation method and application of fish type I collagen-based self-healing composite hydrogel

Abstract

The invention belongs to the technical field of biology and provides a preparation method and application of fish type I collagen-based self-healing composite hydrogel. The method comprises the following steps: taking fish processing byproducts as raw materials, and preparing fish type I collagen by utilizing an enzyme extraction and salting-out column chromatography purification integrated technology. Chitosan is used as a raw material for oxidation, and the oxidized sample is separated and purified to obtain the dialdehyde chitosan. On the basis, the fish type I collagen is used as a base material, genipin is used as a cross-linking agent, chitosan and dialdehyde chitosan are added, and Schiff base reaction is performed to obtain the fish type I collagen based self-healing composite hydrogel. The hydrogel keeps a triple helix structure of fish type I collagen, has good biocompatibility, good self-healing performance, good injection performance and good thermal stability, has good effects of promoting detumescence of frostbite tissues and promoting wound healing, and can be applied to the field of wound dressings.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering, and in particular to a preparation method and application of a fish type I collagen-based self-healing composite hydrogel. Background Art

[0002] Hydrogel is a hydrophilic macromolecular polymer with a three-dimensional network structure. It is porous inside and can hold a large amount of water without dissolving. It has the advantages of good swelling, softness, elasticity, and low surface tension. In recent years, hydrogels have been widely used in many aspects of the biomedical field, especially in tissue engineering and regenerative medicine, drug delivery, stem cell culture and cell therapy. Self-healing composite hydrogel refers to a type of hydrogel whose performance can be restored to or close to the initial state after being damaged by external forces. It is a research hotspot in the hydrogel industry. However, existing self-healing hydrogels have the defect of poor biocompatibility, which restricts their application in the field of high value-added biomedical materials.

[0003] Type I collagen is the main component of the extracellular matrix in animal tissues. It is mainly distributed in tissues such as the epidermis, cornea, and cartilage, and plays a supporting and protective role for the body and organs. Type I collagen maintains a triple helix structure, which is formed by three left-handed helical α peptide chains bonded by hydrogen bonds to form a stable right-handed superhelical structure. The unique triple helix structure of type I collagen makes it have good biocompatibility. Traditional type I collagen comes from terrestrial animals such as pigs and cattle. In recent years, due to the spread of zoonotic diseases around the world, the application of collagen from terrestrial animals has gradually been restricted. Fish type I collagen has become a new source of terrestrial type I collagen substitutes and has become a hot topic of research. However, fish type I collagen hydrogel has technical bottlenecks such as poor self-healing performance and poor thermal stability, which restricts its large-scale production and promotion and application. Therefore, it is urgent to develop a method for preparing and applying a fish type I collagen-based self-healing composite hydrogel that can be industrialized and has good self-healing properties, thermal stability, and biocompatibility, and can promote the detumescence of frostbitten tissue and promote wound healing. Summary of the invention

[0004] The bottleneck problems of the current fish type I collagen hydrogels are poor self-healing performance, poor thermal stability, and inability to achieve large-scale promotion and application. The purpose of the present invention is to provide a method for preparing a fish type I collagen-based self-healing composite hydrogel, which has either an easy-to-control process, a product that maintains the triple helix structure of type I collagen, or has good self-healing performance, or good injection performance, or good thermal stability, or can promote the detumescence of frozen tissue and promote wound healing.

[0005] In order to solve the above technical problems, the technical solution of the present invention is: A method for preparing a fish type I collagen-based composite hydrogel comprises the following steps: (1) Preparation of fish type I collagen Adding water and enzyme catalytic extraction to the pretreated fish processing byproducts, purifying the extract by salting out and column chromatography, and freeze-drying the purified solution to obtain fish type I collagen; (2) Preparation of dialdehyde chitosan Adding an oxidant to the chitosan solution for oxidation, stirring the reaction, stopping the reaction, removing impurities from the reaction product with an ultrafiltration membrane, and freeze-drying the product to obtain dialdehyde chitosan; (3) Preparation of fish type I collagen-based self-healing composite hydrogel The fish type I collagen solution was added into the reaction kettle, chitosan and dialdehyde chitosan were added and stirred evenly, and then a cross-linking agent was added to carry out Schiff base reaction to obtain the fish type I collagen-based self-healing composite hydrogel.

[0006] In some embodiments, water and an enzyme equivalent to about 0.1-1% of the weight of the fish processing by-product feed are added to the pretreated fish processing by-product for catalytic extraction. The enzyme used for extraction can be a protease such as pepsin, but is not limited to pepsin. The pH of the reaction system is 1-2, the reaction temperature is lower than 5°C, and the reaction time is 5-10 hours.

[0007] In some embodiments, in step (1), the fish refers to marine fish or freshwater fish, and the fish processing by-products refer to but are not limited to fish scales, fish skin, fish bladder, and fish bones.

[0008] In some embodiments, in step (1), the salt used for salting out can be sodium chloride, but is not limited to sodium chloride, and the salt added for salting out is about 1-about 5% (W / V).

[0009] In some embodiments, the column chromatography in step (1) refers to but is not limited to ion exchange resin, and the flow rate of the ion exchange resin is about 1-about 10m 3 / h.

[0010] In some embodiments, in step (2), an oxidant is added to the chitosan solution for oxidation, the oxidant may be sodium periodate, the mass ratio of chitosan to the oxidant is about 1:1 to about 5:1, the temperature of the reaction system is about 30 to about 50°C, and the reaction time is about 2 to about 5 hours. In some embodiments, the oxidation is performed by adding sodium periodate for oxidation reaction, stirring the reaction, adding ethylene glycol to terminate the reaction, and removing impurities from the reaction product using an ultrafiltration membrane with a relative molecular mass cutoff of about 1000Da, and freeze-drying to obtain dialdehyde chitosan.

[0011] In some embodiments, in step (3), the mass ratio of fish type I collagen, chitosan and dialdehyde chitosan is about 1:0.8:0.2-about 1:0.2:0.8, and the reaction time is 1-5 hours. In some embodiments, chitosan and dialdehyde chitosan are added to the fish type I collagen solution and stirred evenly, and then a cross-linking agent such as genipin is added to carry out a Schiff base reaction to obtain a fish type I collagen-based self-healing composite hydrogel.

[0012] The above scheme uses fish processing byproducts as raw materials and uses enzyme extraction and salting out column chromatography purification integrated technology to prepare fish type I collagen. Chitosan is used as a raw material, an oxidant is added for oxidation, and the oxidized sample is separated and purified by nanofiltration membrane to obtain dialdehyde chitosan. On this basis, fish type I collagen is used as a substrate, a cross-linking agent is added, and chitosan and dialdehyde chitosan are added in a specific proportion to perform Schiff base reaction to obtain fish type I collagen-based self-healing composite hydrogel. The fish type I collagen-based self-healing composite hydrogel prepared by the present invention maintains the triple helix structure of fish type I collagen, and the distance between the fiber molecular chains of the fish type I collagen-based composite hydrogel is about 1.13nm-about 1.18nm, and the distance between the collagen skeletons is about 0.42nm-about 0.45nm; in the self-healing composite hydrogel, the distance between the collagen fiber molecules is about 1.05nm-about 1.10nm, and the distance between the collagen skeletons is about 0.38nm-about 0.41nm.

[0013] The present invention cross-links fish type I collagen molecules, chitosan molecules and dialdehyde chitosan molecules through dynamic reversible imine bonds and covalent bonds to generate fish type I collagen-based self-healing composite hydrogels. The hydrogels have good self-healing properties, good injection properties, and good thermal stability. They promote the detumescence of frozen tissues and promote wound healing, solving the bottleneck problems of poor self-healing properties and poor thermal stability of collagen-based hydrogels. At the same time, it has good biocompatibility. In summary, the fish type I collagen-based self-healing composite hydrogels prepared by the present invention can be used in the field of wound dressings.

[0014] Compared with the existing technology, it also has the following advantages: The preparation method provided by the present invention forms a fish type I collagen-based self-healing composite hydrogel by cross-linking fish type I collagen molecules, chitosan molecules and dialdehyde chitosan molecules through dynamic reversible imine bonds. The production cost is low, the process is easy to control, and it is suitable for large-scale production. The fish type I collagen-based self-healing composite hydrogel developed by the present invention maintains the triple helix structure of type I collagen and has good biocompatibility. The fish type I collagen-based self-healing composite hydrogel developed by the present invention has good self-healing performance, good injection performance, good thermal stability, and good effects on promoting the detumescence of frostbitten tissue and promoting wound healing, and can be applied in the field of wound dressings. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1In Example 1, the X-ray diffraction pattern of fish type I collagen and self-healing composite hydrogel; Figure 2 In Example 1, the fracture healing experiment of fish type I collagen-based self-healing composite hydrogel, wherein a is the dyed hydrogel sample 1; b is the hydrogel sample 2 dyed with rhodamine B solution; c is the hydrogel samples 1 and 2 put together after cutting; d is the tensile test of the hydrogel after self-healing; Figure 3 In Example 1, fish type Ⅰ collagen-based self-healing composite hydrogel scratch healing experiment, ad are 0h, 0.5h, 1h, 2h self-healing composite hydrogel fracture surface healing diagram; Figure 4 In Example 1, the injectability test of fish type I collagen-based self-healing composite hydrogel Figure 5 In Example 1, the biocompatibility of fish type I collagen-based self-healing composite hydrogel, wherein (a1)-(a3) are cell images of the blank group after 1, 2, and 3 days of the experiment; (b1)-(b3) are cell images of the self-healing composite hydrogel test group after 1, 2, and 3 days of the experiment; Figure 6 In Example 1, fish type Ⅰ collagen-based self-healing composite hydrogel animal experiment, wherein (a1), (a2) and (a3) ​​are wound photos of mice in the blank control group treated for 2, 4, and 7 days; (b1), (b2), and (b3) are wound photos of mice treated for 2, 4, and 7 days with fish type Ⅰ collagen-based self-healing composite hydrogel; Figure 7 In Example 1, the histological manifestations of mouse skin, wherein (a) is normal mouse skin; (b) is blank control group mouse skin; and (c) is fish type I collagen-based self-healing composite hydrogel-treated mouse skin. DETAILED DESCRIPTION

[0016] The technical solution of the present invention is described below by specific examples. It should be understood that the one or more method steps mentioned in the present invention do not exclude the presence of other method steps before and after the combination step or the insertion of other method steps between the steps explicitly mentioned; it should also be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0017] In order to better understand the above technical scheme, the exemplary embodiments of the present invention are described in more detail below. Although exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present invention and to enable the scope of the present invention to be fully communicated to those skilled in the art.

[0018] The present invention is described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and do not limit the present invention in any way.

[0019] Chitosan was purchased from Beijing Solebao Company; Genipin was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; sodium periodate and ethylene glycol were purchased from Sinopharm Chemical Reagent Co., Ltd.; DAPI staining solution and microfilament green fluorescent probe were purchased from Bio-Tech Biotechnology Co., Ltd.; mouse embryonic osteoblast precursor cells (MC3T3-E1) were purchased from Nanjing Kebai Biotechnology Co., Ltd., and ICR male mice (20-22 g) were purchased from Shanghai Slake Laboratory Animal Co., Ltd. Other reagents or equipment were purchased from the market.

[0020] Example 1 The preparation method and application of fish type I collagen-based self-healing composite hydrogel include the following steps: Add 50g of pretreated fish skin to the reactor, add 150g of water, add 0.05g of pepsin, adjust the pH value and control the pH value of the reaction system to 1.5, the reaction temperature is lower than 5°C, and react for 5h to obtain type I collagen crude extract solution. After adding 3% (W / V) sodium chloride to the collagen crude extract solution for salting out, centrifuge it to obtain a precipitate. After adding 0.5M acetic acid to the precipitate and stirring to dissolve it, use ion exchange resin to remove inorganic salts, and the flow rate is 5m 3 / h to obtain type I collagen solution, and freeze-dry the material solution to obtain fish type I collagen.

[0021] The chitosan solution was added to the reaction kettle, and sodium periodate was added to the solution to carry out a composite reaction. In the reaction system, the mass ratio of chitosan to sodium periodate was 1.5:1, the temperature of the reaction system was 40°C, and the reaction time was 3h. The reaction product was separated and purified using an ultrafiltration membrane with a relative molecular mass cutoff of 1000Da, the temperature of the solution system was controlled below 5°C, and the material solution after membrane separation was freeze-dried to obtain dialdehyde chitosan.

[0022] The fish type I collagen solution was added into the reactor, chitosan and dialdehyde chitosan were added and stirred evenly, then the cross-linking agent genipin was added to carry out the Schiff base reaction. In the reaction system, the mass ratio of fish type I collagen, chitosan and dialdehyde chitosan was 1:0.6:0.4. The reaction was carried out for 2 hours to obtain the fish type I collagen-based self-healing composite hydrogel.

[0023] [Structural characterization] The structure of the fish type I collagen and fish type I collagen-based self-healing composite hydrogel prepared in this embodiment was detected by X-ray diffraction. The test results showed that the distance between the fiber molecular chains of fish type I collagen was 1.16nm, and the distance between the collagen skeletons was 0.44nm; in the self-healing composite hydrogel, the distance between the collagen fiber molecules was 1.09nm, and the distance between the collagen skeletons was 0.41nm. The distance between the fiber molecules and the distance between the skeletons of the self-healing composite hydrogel collagen were lower than those of fish type I collagen, indicating that the fish type I collagen molecules, chitosan molecules and dialdehyde chitosan molecules changed the crystallinity and molecular arrangement conformation of the hydrogel through the imine bond cross-linking reaction, making the distance between the collagen molecules and the skeleton closer, but did not destroy the triple helix structure of the collagen. ( Figure 1 ) [Characterization of physical and chemical properties] The thermal denaturation temperature (T d ) to characterize the thermal stability of the sample. d The T of the fish type I collagen-based self-healing composite hydrogel prepared in this example was d The T of the self-healing composite hydrogel is 37.5℃. d It is 11.8℃ higher than that of fish type I collagen, indicating that the cross-linking of fish type I collagen molecules, chitosan molecules and dialdehyde chitosan molecules through imine bonds makes the intermolecular intercalation tighter and improves the thermal stability of the product.

[0024] [Self-healing performance] The fish type I collagen-based self-healing composite hydrogel was made into two circular hydrogels, one of which was not dyed and the other was dyed with rhodamine B solution. The two hydrogels were cut into two halves, and the two different semicircular hydrogels were cut together to observe their self-healing composite. After 2 hours, they were picked up with tweezers and manually stretched to judge the self-healing properties of the hydrogel. The experimental results show that after 2 hours, the fish type I collagen-based self-healing composite hydrogel has good tensile strength and shows good self-healing properties. The dynamic reversible imine bond of the fish type I collagen-based self-healing composite hydrogel gives it good self-healing properties ( Figure 2 ) After the fish type I collagen-based self-healing composite hydrogel was scratched, it was placed under an inverted microscope to record the changes in the scratches at different time periods. The experimental results showed that the scratches on the fish type I collagen-based self-healing composite hydrogel were blurred after 2 hours, and the self-healing effect was good. This was due to the re-generation of dynamic imine bonds by the rearrangement of the internal molecules of the hydrogel sample. ( Figure 3 ) [Injection performance] A disposable syringe equipped with a 21G needle was used to inject the fish type I collagen-based self-healing composite hydrogel and write the letters HQU. The experimental results showed that the injected hydrogel had uniform lines and good injection performance. Figure 4 ) [Degradation performance] Prepare 3 cylindrical hydrogels of size (Φ30mm, h=4mm). Immerse the fish type I collagen-based self-healing composite hydrogel in a beaker containing 15mL, 1U / mL collagenase-PBS solution. Then, place it in a drying oven at 37°C. The collagenase-PBS solution was changed once a day, and the hydrogel was taken out at the specified time point, rinsed with deionized water, and the surface moisture of the fish type I collagen-based self-healing composite hydrogel was removed and weighed. The calculation formula for the in vitro degradation rate of the fish type I collagen-based self-healing composite hydrogel is as follows: Formula (1) Where: W0 represents the mass of the hydrogel before degradation; W t Indicates the mass of the hydrogel after degradation The experimental results show that the equilibrium degradation rate of fish type I collagen-based self-healing composite hydrogel is 87.94%. The hydrogel has good degradability and can avoid secondary damage caused by dressing changes when used as a wound dressing.

[0025] [Biocompatibility] MC3T3-E1 was inoculated on the surface of fish type I collagen-based self-healing composite hydrogel to evaluate the biocompatibility of the self-healing composite hydrogel. In this experiment, DAPI staining solution and microfilament green fluorescent probe were used to stain the nucleus and cytoplasm of MC3T3-E1 cells, respectively. The experimental results showed that the nucleus was stained blue, the cytoplasm was green, the cells were spindle-shaped, and longer tentacles were observed in the cells inoculated on the surface of the hydrogel, indicating that the cells grew well on the surface of the hydrogel. From the cell distribution on the second and third days of each experimental group, it can be seen that the cells inoculated on the surface of the hydrogel have better diffusivity than the blank control group. In addition, the cells in the experimental group can adhere well to the surface of the collagen-based hydrogel and grow, showing good cell adhesion; from the cell density, it can be seen that the collagen-based hydrogel has good cell compatibility. In short, the fish type I collagen-based self-healing composite hydrogel has good biocompatibility and adhesion to cells. It has a positive effect on promoting wound repair and has potential application prospects in wound dressings. ( Figure 5 ) [Animal Experiments] This experiment used a mouse frostbite model to study the ability of fish type I collagen-based self-healing composite hydrogel as a wound dressing to promote wound healing. The experiment showed that the frostbite site of the mouse began to become red and swollen the day after modeling. After 7 consecutive days of drug treatment, compared with the blank control group, the wounds of the mice in the fish type I collagen-based self-healing composite hydrogel test group showed obvious swelling reduction and better healing effect, while the wounds of the mice in the blank control group were still obvious ( Figure 6 ).

[0026] The swelling value and swelling inhibition rate were used to quantitatively evaluate the therapeutic effects of the two hydrogels.

[0027] Formula (2) The experimental results showed that the swelling inhibition rate of the fish type I collagen-based self-healing composite hydrogel was 80.5%. The hydrogels all had good therapeutic effects, and the experimental results were consistent with the wound conditions, indicating that the hydrogel developed in this study can be used as wound dressings in the biomedical field.

[0028] The tissues around the wounds and normal tissues of mice were taken for histological analysis. The tissue sections were fixed with 10% neutral buffered formalin fixative, dehydrated in graded sucrose, embedded in paraffin, and frozen in liquid nitrogen. A cryostat was used to obtain 10um continuous cryosections. After staining with hematoxylin and eosin, the sections were observed with an inverted microscope. The experimental results showed that the skin of mice in the blank control group had obvious edema, epidermal defects, a large number of inflammatory cells in the dermis, disappearance of hair follicles and sebaceous glands, and damage to the subcutaneous tissue and muscle layer. The number of inflammatory cells in the skin of mice treated with fish type I collagen-based self-healing composite hydrogel was greatly reduced, the subcutaneous tissue and muscle layer were restored to a great extent, the degree of epidermal repair was close to that of normal skin, and skin appendages such as hair follicles and sebaceous glands were formed in the dermis, indicating that fish type I collagen-based self-healing composite hydrogel has the effect of promoting skin tissue repair ( Figure 7 ).

[0029] Example 2 The preparation method and application of fish type I collagen-based self-healing composite hydrogel include the following steps: Add 10g of pretreated fish maw to the reactor, add 100g of water, add 0.05g of pepsin, adjust the pH value and control the pH value of the reaction system to 2, the reaction temperature is lower than 5°C, and react for 6h to obtain type I collagen crude extract solution. After adding 1% (W / V) sodium chloride to the collagen crude extract solution for salting out, centrifuge it to obtain a precipitate. After adding 0.5M acetic acid to the precipitate and stirring to dissolve it, use ion exchange resin to remove inorganic salts, and the flow rate is 1m 3 / h to obtain type I collagen solution, and freeze-dry the material solution to obtain fish type I collagen.

[0030] The chitosan solution was added to the reaction kettle, and sodium periodate was added to the solution to carry out a composite reaction. In the reaction system, the mass ratio of chitosan to sodium periodate was 1:1, the temperature of the reaction system was 30°C, and the reaction time was 2h. The reaction product was separated and purified using an ultrafiltration membrane with a cutoff relative molecular mass of 1000Da, the temperature of the solution system was controlled below 5°C, and the material solution after membrane separation was freeze-dried to obtain dialdehyde chitosan.

[0031] The fish type I collagen solution was added to the reactor, chitosan and dialdehyde chitosan were added and stirred evenly, and then the cross-linking agent genipin was added to carry out the Schiff base reaction. In the reaction system, the mass ratio of fish type I collagen, chitosan and dialdehyde chitosan was 1:0.8:0.2. The reaction was carried out for 1 hour to obtain the fish type I collagen-based self-healing composite hydrogel. [Structural characterization] The structures of the fish type I collagen and the fish type I collagen-based self-healing composite hydrogel prepared in this embodiment were detected by X-ray diffraction. The detection results showed that the distance between the fiber molecular chains of fish type I collagen was 1.18 nm, and the distance between the collagen skeletons was 0.45 nm; in the self-healing composite hydrogel, the distance between the collagen fiber molecules was 1.10 nm, and the distance between the collagen skeletons was 0.39 nm. The distance between the fiber molecules and the distance between the skeletons of the self-healing composite hydrogel collagen were both lower than those of fish type I collagen, indicating that the fish type I collagen molecules, chitosan molecules and dialdehyde chitosan molecules changed the crystallinity and molecular arrangement conformation of the hydrogel through the imine bond cross-linking reaction, making the distance between the collagen molecules and the skeleton closer, but without destroying the triple helix structure of the collagen.

[0032] [Characterization of physical and chemical properties] The thermal denaturation temperature (T d ) to characterize the thermal stability of the sample. d The T of the fish type I collagen-based self-healing composite hydrogel prepared in this example was 28.4°C. d The T of the self-healing composite hydrogel is 39.2℃. d It is 10.8℃ higher than that of fish type I collagen, indicating that the cross-linking of fish type I collagen molecules, chitosan molecules and dialdehyde chitosan molecules through imine bonds makes the intermolecular intercalation tighter and improves the thermal stability of the product.

[0033] [Self-healing performance] The fish type I collagen-based self-healing composite hydrogel was made into two circular hydrogels, one of which was not stained and the other was stained with rhodamine B solution. The two hydrogels were cut into two halves, and the two different semicircular hydrogels were cut together to observe their self-healing properties. After 2 hours, they were picked up with tweezers and manually stretched to judge the self-healing properties of the hydrogel. The experimental results show that after 2 hours, the fish type I collagen-based self-healing composite hydrogel has good tensile strength and shows good self-healing properties. The dynamic reversible imine bond of the fish type I collagen-based self-healing composite hydrogel gives it good self-healing properties After the fish type I collagen-based self-healing composite hydrogel was scratched, it was placed under an inverted microscope to record the changes in the scratches at different time periods. The experimental results showed that the scratches on the fish type I collagen-based self-healing composite hydrogel were blurred after 2 hours, and the self-healing effect was good. This was due to the re-generation of dynamic imine bonds by molecular rearrangement inside the hydrogel sample.

[0034] [Injection performance] A disposable syringe equipped with a "21G" sized needle was used to inject the fish type I collagen-based self-healing composite hydrogel and write the letters HQU. The experimental results showed that the injected hydrogel had uniform lines and good injection performance.

[0035] [Degradation performance] Prepare 3 cylindrical hydrogels of size (Φ30mm, h=4mm). Immerse the fish type I collagen-based self-healing composite hydrogel in a beaker containing 15mL, 1U / mL collagenase-PBS solution. Then, place it in a drying oven at 37°C. The collagenase-PBS solution was changed once a day, and the hydrogel was taken out at the specified time point, rinsed with deionized water, and the surface moisture of the fish type I collagen-based self-healing composite hydrogel was removed and weighed. The calculation formula for the in vitro degradation rate of the fish type I collagen-based self-healing composite hydrogel is as follows: Formula (3) Where: W0 represents the mass of the hydrogel before degradation; W t Indicates the mass of the hydrogel after degradation The experimental results show that the equilibrium degradation rate of fish type I collagen-based self-healing composite hydrogel is 86.29%. The hydrogel has good degradability and can avoid secondary damage caused by dressing changes when used as a wound dressing.

[0036] [Biocompatibility] MC3T3-E1 was inoculated on the surface of fish type I collagen-based self-healing composite hydrogel to evaluate the biocompatibility of the self-healing composite hydrogel. In this experiment, DAPI staining solution and microfilament green fluorescent probe were used to stain the nucleus and cytoplasm of MC3T3-E1 cells, respectively. The experimental results showed that the nucleus was stained blue, the cytoplasm was green, the cells were spindle-shaped, and longer tentacles were observed in the cells inoculated on the surface of the hydrogel, indicating that the cells grew well on the surface of the hydrogel. From the cell distribution on the second and third days of each experimental group, it can be seen that the cells inoculated on the surface of the hydrogel have better diffusivity than the blank control group. In addition, the cells in the experimental group can adhere well to the surface of the collagen-based hydrogel and grow, showing good cell adhesion; from the cell density, it can be seen that the collagen-based hydrogel has good cell compatibility. In short, the fish type I collagen-based self-healing composite hydrogel has good biocompatibility and adhesion to cells. It has a positive effect on promoting wound repair and has potential application prospects in wound dressings.

[0037] [Animal Experiments] This experiment used a mouse frostbite model to study the ability of fish type I collagen-based self-healing composite hydrogel as a wound dressing to promote wound healing. The experiment showed that redness and swelling began to appear on the frostbite site of the mouse the day after modeling. After 7 consecutive days of drug treatment, compared with the blank control group, the wounds of the mice in the fish type I collagen-based self-healing composite hydrogel test group showed obvious swelling reduction and better healing effect, while the wounds of the mice in the blank control group were still relatively obvious.

[0038] The swelling value and swelling inhibition rate were used to quantitatively evaluate the therapeutic effects of the two hydrogels.

[0039] Formula (4) The experimental results showed that the swelling inhibition rate of the fish type I collagen-based self-healing composite hydrogel was 85.3%. The hydrogels all had good therapeutic effects, and the experimental results were consistent with the wound conditions, indicating that the hydrogel developed in this study can be used as wound dressings in the biomedical field.

[0040] The tissues around the wound and normal tissues of mice were taken for histological analysis. The tissue sections were fixed with 10% neutral buffered formalin fixative, dehydrated in graded sucrose, embedded in paraffin, and frozen in liquid nitrogen. A cryostat was used to obtain 10um continuous cryosections. After staining with hematoxylin and eosin, the sections were observed with an inverted microscope. The experimental results showed that the skin of mice in the blank control group had obvious edema, epidermal defects, a large number of inflammatory cells in the dermis, disappearance of hair follicles and sebaceous glands, and damage to the subcutaneous tissue and muscle layer. The number of inflammatory cells in the skin of mice treated with fish type I collagen-based self-healing composite hydrogel was greatly reduced, the subcutaneous tissue and muscle layer were restored to a great extent, the degree of epidermal repair was close to that of normal skin, and skin appendages such as hair follicles and sebaceous glands were formed in the dermis, indicating that fish type I collagen-based self-healing composite hydrogel has the effect of promoting skin tissue repair.

[0041] Example 3 The preparation method and application of fish type I collagen-based self-healing composite hydrogel include the following steps: Add 100g of pretreated fish scales to the reactor, add 500g of water, add 0.8g of pepsin, adjust the pH value and control the pH value of the reaction system to 1, the reaction temperature is lower than 5°C, react for 8h, and obtain type I collagen crude extract solution. After adding 5% (W / V) sodium chloride to the collagen crude extract solution for salting out, centrifuge it to obtain a precipitate. After adding 0.5M acetic acid to the precipitate and stirring to dissolve it, use ion exchange resin to remove inorganic salts, and the flow rate is 8m 3 / h to obtain type I collagen solution, and freeze-dry the material solution to obtain fish type I collagen.

[0042] The chitosan solution was added to the reaction kettle, and sodium periodate was added to the solution to carry out a composite reaction. In the reaction system, the mass ratio of chitosan to sodium periodate was 5:1, the temperature of the reaction system was 40°C, and the reaction time was 4h. The reaction product was separated and purified using an ultrafiltration membrane with a relative molecular mass cutoff of 1000Da, the temperature of the solution system was controlled below 5°C, and the material solution after membrane separation was freeze-dried to obtain dialdehyde chitosan.

[0043] The fish type I collagen solution was added to the reactor, chitosan and dialdehyde chitosan were added and stirred evenly, and then the cross-linking agent genipin was added to carry out the Schiff base reaction. In the reaction system, the mass ratio of fish type I collagen, chitosan and dialdehyde chitosan was 1:0.5:0.5. The reaction was carried out for 3 hours to obtain fish type I collagen-based self-healing composite hydrogel. [Structural characterization] The structures of the fish type I collagen and the fish type I collagen-based self-healing composite hydrogel prepared in this embodiment were detected by X-ray diffraction. The detection results showed that the distance between the fiber molecular chains of fish type I collagen was 1.13 nm, and the distance between the collagen skeletons was 0.42 nm; in the self-healing composite hydrogel, the distance between the collagen fiber molecules was 1.07 nm, and the distance between the collagen skeletons was 0.40 nm. The distance between the fiber molecules and the distance between the skeletons of the self-healing composite hydrogel collagen were both lower than those of fish type I collagen, indicating that the fish type I collagen molecules, chitosan molecules and dialdehyde chitosan molecules changed the crystallinity and molecular arrangement conformation of the hydrogel through the imine bond cross-linking reaction, making the distance between the collagen molecules and the skeleton closer, but without destroying the triple helix structure of the collagen.

[0044] [Characterization of physical and chemical properties] The thermal denaturation temperature (T d ) to characterize the thermal stability of the sample. d The T of the fish type I collagen-based self-healing composite hydrogel prepared in this example was d The T of the self-healing composite hydrogel is 40.3℃. d It is 11℃ higher than that of fish type I collagen, indicating that the cross-linking of fish type I collagen molecules, chitosan molecules and dialdehyde chitosan molecules through imine bonds makes the intermolecular intercalation tighter and improves the thermal stability of the product.

[0045] [Self-healing performance] The fish type I collagen-based self-healing composite hydrogel was made into two circular hydrogels, one of which was not stained and the other was stained with rhodamine B solution. The two hydrogels were cut into two halves, and the two different semicircular hydrogels were cut together to observe their self-healing properties. After 2 hours, they were picked up with tweezers and manually stretched to judge the self-healing properties of the hydrogel. The experimental results show that after 2 hours, the fish type I collagen-based self-healing composite hydrogel has good tensile strength and shows good self-healing properties. The dynamic reversible imine bond of the fish type I collagen-based self-healing composite hydrogel gives it good self-healing properties After the fish type I collagen-based self-healing composite hydrogel was scratched, it was placed under an inverted microscope to record the changes in the scratches at different time periods. The experimental results showed that the scratches on the fish type I collagen-based self-healing composite hydrogel were blurred after 2 hours, and the self-healing effect was good. This was due to the re-generation of dynamic imine bonds by molecular rearrangement inside the hydrogel sample.

[0046] [Injection performance] A disposable syringe equipped with a "21G" sized needle was used to inject the fish type I collagen-based self-healing composite hydrogel and write the letters HQU. The experimental results showed that the injected hydrogel had uniform lines and good injection performance.

[0047] [Degradation performance] Prepare 3 cylindrical hydrogels of size (Φ30mm, h=4mm). Immerse the fish type I collagen-based self-healing composite hydrogel in a beaker containing 15mL, 1U / mL collagenase-PBS solution. Then, place it in a drying oven at 37°C. The collagenase-PBS solution was changed once a day, and the hydrogel was taken out at the specified time point, rinsed with deionized water, and the surface moisture of the fish type I collagen-based self-healing composite hydrogel was removed and weighed. The calculation formula for the in vitro degradation rate of the fish type I collagen-based self-healing composite hydrogel is as follows: Formula (5) Where: W0 represents the mass of the hydrogel before degradation; W t Indicates the mass of the hydrogel after degradation The experimental results show that the equilibrium degradation rate of fish type I collagen-based self-healing composite hydrogel is 82.46%. The hydrogel has good degradability and can avoid secondary damage caused by dressing changes when used as a wound dressing.

[0048] [Biocompatibility] MC3T3-E1 was inoculated on the surface of fish type I collagen-based self-healing composite hydrogel to evaluate the biocompatibility of the self-healing composite hydrogel. In this experiment, DAPI staining solution and microfilament green fluorescent probe were used to stain the nucleus and cytoplasm of MC3T3-E1 cells, respectively. The experimental results showed that the nucleus was stained blue, the cytoplasm was green, the cells were spindle-shaped, and longer tentacles were observed in the cells inoculated on the surface of the hydrogel, indicating that the cells grew well on the surface of the hydrogel. From the cell distribution on the second and third days of each experimental group, it can be seen that the cells inoculated on the surface of the hydrogel have better diffusivity than the blank control group. In addition, the cells in the experimental group can adhere well to the surface of the collagen-based hydrogel and grow, showing good cell adhesion; from the cell density, it can be seen that the collagen-based hydrogel has good cell compatibility. In short, the fish type I collagen-based self-healing composite hydrogel has good biocompatibility and adhesion to cells. It has a positive effect on promoting wound repair and has potential application prospects in wound dressings.

[0049] [Animal Experiments] This experiment used a mouse frostbite model to study the ability of fish type I collagen-based self-healing composite hydrogel as a wound dressing to promote wound healing. The experiment showed that redness and swelling began to appear on the frostbite site of the mouse the day after modeling. After 7 consecutive days of drug treatment, compared with the blank control group, the wounds of the mice in the fish type I collagen-based self-healing composite hydrogel test group showed obvious swelling reduction and better healing effect, while the wounds of the mice in the blank control group were still relatively obvious.

[0050] The swelling value and swelling inhibition rate were used to quantitatively evaluate the therapeutic effects of the two hydrogels.

[0051] Formula (6) The experimental results showed that the swelling inhibition rate of the fish type I collagen-based self-healing composite hydrogel was 83.69%. The hydrogels all had good therapeutic effects, and the experimental results were consistent with the wound conditions, indicating that the hydrogel developed in this study can be used as wound dressings in the biomedical field.

[0052] The tissues around the wound and normal tissues of mice were taken for histological analysis. The tissue sections were fixed with 10% neutral buffered formalin fixative, dehydrated in graded sucrose, embedded in paraffin, and frozen in liquid nitrogen. A cryostat was used to obtain 10um continuous cryosections. After staining with hematoxylin and eosin, the sections were observed with an inverted microscope. The experimental results showed that the skin of mice in the blank control group had obvious edema, epidermal defects, a large number of inflammatory cells in the dermis, disappearance of hair follicles and sebaceous glands, and damage to the subcutaneous tissue and muscle layer. The number of inflammatory cells in the skin of mice treated with fish type I collagen-based self-healing composite hydrogel was greatly reduced, the subcutaneous tissue and muscle layer were restored to a great extent, the degree of epidermal repair was close to that of normal skin, and skin appendages such as hair follicles and sebaceous glands were formed in the dermis, indicating that fish type I collagen-based self-healing composite hydrogel has the effect of promoting skin tissue repair.

[0053] Example 4 The preparation method and application of fish type I collagen-based self-healing composite hydrogel include the following steps: Add 200g of pretreated fish bones to the reactor, add 600g of water, add 0.2g of pepsin, adjust the pH value and control the pH value of the reaction system to 1, the reaction temperature is lower than 5°C, react for 10h, and obtain type I collagen crude extract solution. After adding 4% (W / V) sodium chloride to the collagen crude extract solution for salting out, centrifuge it to obtain a precipitate. After adding 0.5M acetic acid to the precipitate and stirring to dissolve it, use ion exchange resin to remove inorganic salts, and the flow rate is 10m 3 / h to obtain type I collagen solution, and freeze-dry the material solution to obtain fish type I collagen.

[0054] The chitosan solution was added to the reaction kettle, and sodium periodate was added to the solution to carry out a composite reaction. In the reaction system, the mass ratio of chitosan to sodium periodate was 3:1, the temperature of the reaction system was 50°C, and the reaction time was 5h. The reaction product was separated and purified using an ultrafiltration membrane with a relative molecular mass cutoff of 1000Da, the temperature of the solution system was controlled below 5°C, and the material solution after membrane separation was freeze-dried to obtain dialdehyde chitosan.

[0055] The fish type I collagen solution was added to the reactor, chitosan and dialdehyde chitosan were added and stirred evenly, and then the cross-linking agent genipin was added to carry out the Schiff base reaction. In the reaction system, the mass ratio of fish type I collagen, chitosan and dialdehyde chitosan was 1:0.2:0.8. The reaction was carried out for 5 hours to obtain fish type I collagen-based self-healing composite hydrogel. [Structural characterization] The structures of the fish type I collagen and the fish type I collagen-based self-healing composite hydrogel prepared in this embodiment were detected by X-ray diffraction. The detection results showed that the distance between the fiber molecular chains of fish type I collagen was 1.15 nm, and the distance between the collagen skeletons was 0.43 nm; in the self-healing composite hydrogel, the distance between the collagen fiber molecules was 1.05 nm, and the distance between the collagen skeletons was 0.38 nm. The distance between the fiber molecules and the distance between the skeletons of the self-healing composite hydrogel collagen were both lower than those of fish type I collagen, indicating that the fish type I collagen molecules, chitosan molecules and dialdehyde chitosan molecules changed the crystallinity and molecular arrangement conformation of the hydrogel through the imine bond cross-linking reaction, making the distance between the collagen molecules and the skeleton closer, but without destroying the triple helix structure of the collagen.

[0056] [Characterization of physical and chemical properties] The thermal denaturation temperature (T d ) to characterize the thermal stability of the sample. d The T of the fish type I collagen-based self-healing composite hydrogel prepared in this example was d The T of the self-healing composite hydrogel is 40.5℃. d It is 10.7℃ higher than that of fish type I collagen, indicating that the cross-linking of fish type I collagen molecules, chitosan molecules and dialdehyde chitosan molecules through imine bonds makes the intermolecular intercalation tighter and improves the thermal stability of the product.

[0057] [Self-healing performance] The fish type I collagen-based self-healing composite hydrogel was made into two circular hydrogels, one of which was not stained and the other was stained with rhodamine B solution. The two hydrogels were cut into two halves, and the two different semicircular hydrogels were cut together to observe their self-healing properties. After 2 hours, they were picked up with tweezers and manually stretched to judge the self-healing properties of the hydrogel. The experimental results show that after 2 hours, the fish type I collagen-based self-healing composite hydrogel has good tensile strength and shows good self-healing properties. The dynamic reversible imine bond of the fish type I collagen-based self-healing composite hydrogel gives it good self-healing properties After the fish type I collagen-based self-healing composite hydrogel was scratched, it was placed under an inverted microscope to record the changes in the scratches at different time periods. The experimental results showed that the scratches on the fish type I collagen-based self-healing composite hydrogel were blurred after 2 hours, and the self-healing effect was good. This was due to the re-generation of dynamic imine bonds by molecular rearrangement inside the hydrogel sample.

[0058] [Injection performance] A disposable syringe equipped with a "21G" sized needle was used to inject the fish type I collagen-based self-healing composite hydrogel and write the letters HQU. The experimental results showed that the injected hydrogel had uniform lines and good injection performance.

[0059] [Degradation performance] Prepare 3 cylindrical hydrogels of size (Φ30mm, h=4mm). Immerse the fish type I collagen-based self-healing composite hydrogel in a beaker containing 15mL, 1U / mL collagenase-PBS solution. Then, place it in a drying oven at 37°C. The collagenase-PBS solution was changed once a day, and the hydrogel was taken out at the specified time point, rinsed with deionized water, and the surface moisture of the fish type I collagen-based self-healing composite hydrogel was removed and weighed. The calculation formula for the in vitro degradation rate of the fish type I collagen-based self-healing composite hydrogel is as follows: Formula (7) Where: W0 represents the mass of the hydrogel before degradation; W t Indicates the mass of the hydrogel after degradation The experimental results show that the equilibrium degradation rate of fish type I collagen-based self-healing composite hydrogel is 81.57%. The hydrogel has good degradability and can avoid secondary damage caused by dressing changes when used as a wound dressing.

[0060] [Biocompatibility] MC3T3-E1 was inoculated on the surface of fish type I collagen-based self-healing composite hydrogel to evaluate the biocompatibility of the self-healing composite hydrogel. In this experiment, DAPI staining solution and microfilament green fluorescent probe were used to stain the nucleus and cytoplasm of MC3T3-E1 cells, respectively. The experimental results showed that the nucleus was stained blue, the cytoplasm was green, the cells were spindle-shaped, and longer tentacles were observed in the cells inoculated on the surface of the hydrogel, indicating that the cells grew well on the surface of the hydrogel. From the cell distribution on the second and third days of each experimental group, it can be seen that the cells inoculated on the surface of the hydrogel have better diffusivity than the blank control group. In addition, the cells in the experimental group can adhere well to the surface of the collagen-based hydrogel and grow, showing good cell adhesion; from the cell density, it can be seen that the collagen-based hydrogel has good cell compatibility. In short, the fish type I collagen-based self-healing composite hydrogel has good biocompatibility and adhesion to cells. It has a positive effect on promoting wound repair and has potential application prospects in wound dressings.

[0061] [Animal Experiments] This experiment used a mouse frostbite model to study the ability of fish type I collagen-based self-healing composite hydrogel as a wound dressing to promote wound healing. The experiment showed that redness and swelling began to appear on the frostbite site of the mouse the day after modeling. After 7 consecutive days of drug treatment, compared with the blank control group, the wounds of the mice in the fish type I collagen-based self-healing composite hydrogel test group showed obvious swelling reduction and better healing effect, while the wounds of the mice in the blank control group were still relatively obvious.

[0062] The swelling value and swelling inhibition rate were used to quantitatively evaluate the therapeutic effects of the two hydrogels.

[0063] Formula (8) The experimental results showed that the swelling inhibition rate of the fish type I collagen-based self-healing composite hydrogel was 82.57%. The hydrogels all had good therapeutic effects, and the experimental results were consistent with the wound conditions, indicating that the hydrogels developed in this study can be used as wound dressings in the biomedical field.

[0064] The tissues around the wound and normal tissues of mice were taken for histological analysis. The tissue sections were fixed with 10% neutral buffered formalin fixative, dehydrated in graded sucrose, embedded in paraffin, and frozen in liquid nitrogen. A cryostat was used to obtain 10um continuous cryosections. After staining with hematoxylin and eosin, the sections were observed with an inverted microscope. The experimental results showed that the skin of mice in the blank control group had obvious edema, epidermal defects, a large number of inflammatory cells in the dermis, disappearance of hair follicles and sebaceous glands, and damage to the subcutaneous tissue and muscle layer. The number of inflammatory cells in the skin of mice treated with fish type I collagen-based self-healing composite hydrogel was greatly reduced, the subcutaneous tissue and muscle layer were restored to a great extent, the degree of epidermal repair was close to that of normal skin, and skin appendages such as hair follicles and sebaceous glands were formed in the dermis, indicating that fish type I collagen-based self-healing composite hydrogel has the effect of promoting skin tissue repair.

Claims

1. A method for preparing a fish type I collagen-based composite hydrogel, comprising the following steps: (1) Preparation of fish type I collagen Adding water and enzyme catalytic extraction to the pretreated fish processing byproducts, purifying the extract by salting out and column chromatography, and freeze-drying the purified solution to obtain fish type I collagen; (2) Preparation of dialdehyde chitosan Adding an oxidant to the chitosan solution for oxidation, stirring the reaction, stopping the reaction, removing impurities from the reaction product with an ultrafiltration membrane, and freeze-drying the product to obtain dialdehyde chitosan; (3) Preparation of fish type I collagen-based self-healing composite hydrogel The fish type I collagen solution was added into the reaction kettle, chitosan and dialdehyde chitosan were added and stirred evenly, and then a cross-linking agent was added to carry out Schiff base reaction to obtain the fish type I collagen-based self-healing composite hydrogel.

2. The method for preparing the fish type I collagen-based composite hydrogel according to claim 1, characterized in that: In step (3), the mass ratio of fish type I collagen, chitosan and dialdehyde chitosan is 1:0.8:0.2-1:0.2:0.

8.

3. The method for preparing the fish type I collagen-based composite hydrogel according to claim 1, characterized in that: In step (1), the enzyme used for extraction is but not limited to pepsin, the pH of the reaction system is 1-2, the reaction temperature is lower than 5°C, and the reaction time is 5-10 hours.

4. The method for preparing the fish type I collagen-based composite hydrogel according to claim 1, characterized in that: In step (1), the salt used for salting out is selected from sodium chloride, and the salt added for salting out is 1-5% (W / V).

5. The method for preparing the fish type I collagen-based composite hydrogel according to claim 1, characterized in that: In step (1), column chromatography refers to but is not limited to ion exchange resin, and the flow rate of the ion exchange resin is 1-10m 3 / h.

6. The method for preparing the fish type I collagen-based composite hydrogel according to claim 1, characterized in that: In step (2), the mass ratio of chitosan to oxidant in the reaction is 1:1-5:

1.

7. The method for preparing the fish type I collagen-based composite hydrogel according to claim 1, characterized in that: The oxidation in step (2) is performed by adding sodium periodate, stirring the reaction, and then adding ethylene glycol to terminate the reaction. The reaction product is removed from impurities by an ultrafiltration membrane with a relative molecular mass cutoff of 1000Da, and then freeze-dried to obtain dialdehyde chitosan.

8. The method for preparing the fish type I collagen-based composite hydrogel according to claim 1, characterized in that: The cross-linking agent in step (3) is selected from genipin.

9. The fish type I collagen-based composite hydrogel according to claim 1, characterized in that: The distance between fiber molecular chains of the fish type I collagen-based composite hydrogel is 1.13nm-1.18nm, and the distance between collagen skeletons is 0.42nm-0.45nm; in the self-healing composite hydrogel, the distance between collagen fiber molecules is 1.05nm-1.10nm, and the distance between collagen skeletons is 0.38nm-0.41nm.

10. Use of the fish type I collagen-based composite hydrogel prepared by any one of the preparation methods of claims 1 to 7 or the fish type I collagen-based composite hydrogel according to claim 8 in the preparation of wound dressings.

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