CN116570759A - Method for constructing semi-interpenetrating network bioactive hydrogel through secondary molding and product and application of product - Google Patents
Method for constructing semi-interpenetrating network bioactive hydrogel through secondary molding and product and application of product 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
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0061—Use of materials characterised by their function or physical properties
- A61L26/008—Hydrogels or hydrocolloids
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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
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
- A61L26/0019—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
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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
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
- A61L26/0023—Polysaccharides
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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
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0061—Use of materials characterised by their function or physical properties
- A61L26/0066—Medicaments; Biocides
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- General Health & Medical Sciences (AREA)
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Abstract
The invention discloses a method for constructing semi-interpenetrating network bioactive hydrogel by secondary molding, a product and application of the product, and belongs to the technical field of polymer gel. The invention designs a method for constructing semi-interpenetrating network bioactive hydrogel by secondary molding, which comprises the steps of forming a rigid molecular skeleton by chemical crosslinking of cellulose and polyglutamic acid, inserting third polymer polylysine into the molecular skeleton to obtain hydrogel, and accurately designing parameters such as types, dosages and the like of the three polymers. The hydrogel prepared by the method has excellent water absorbability, biocompatibility, mechanical property and antibacterial property, is suitable for repairing various skin injuries when applied to hydrogel dressing, and has good clinical transformation prospect.
Description
Technical Field
The invention relates to the technical field of polymer gel, in particular to a method for constructing semi-interpenetrating network bioactive hydrogel by secondary molding, a product thereof and application of the product.
Background
The hydrogel dressing is a wound dressing with great prospect, has a network structure composed of hydrophilic substances and has good water absorption. The medical hydrogel material is favorable for promoting wound healing, has good biocompatibility, and is safe and environment-friendly. The existing hydrogel dressing has some common defects and difficulties. For example, most antimicrobial hydrogel dressings are loaded with antimicrobial agents such as antibiotics, metal, and oxide nanoparticles. They are extremely prone to spread into the wound microenvironment, leading to bacterial resistance, pigmentation and other derived problems. On the other hand, the hydrogel texture is soft, its ability to adapt is extremely sensitive to cracks, and it is difficult to closely conform to the skin, especially at the joint site of exercise.
Chinese patent CN115531296a discloses a drug-loaded polyglutamic acid based hydrogel and a preparation method thereof, comprising the following steps: a) Dissolving polyglutamic acid in water to obtain polyglutamic acid; b) Dissolving colchicine powder in an organic solvent to obtain colchicine solution; c) Dissolving polylysine in water to obtain polylysine solution; d) And mixing the polyglutamic acid and colchicine solution with the polylysine solution, and standing to obtain the drug-loaded polyglutamate hydrogel. Chinese patent CN103656729a provides a hydrogel based on a cross-linked polymer of gamma-polyglutamic acid and epsilon-polylysine and a preparation method thereof, wherein the hydrogel is prepared by cross-linking gamma-polyglutamic acid and epsilon-polylysine by a cross-linking agent. The preparation principle of the hydrogel formed at one time is that the active antibacterial group amino on the polylysine is generally utilized to carry out chemical crosslinking reaction with other groups to obtain the hydrogel, and the preparation method not only can make the polylysine difficult to release to a wound area, but also can consume the antibacterial group amino through chemical crosslinking, so that the antibacterial activity of the polylysine is greatly weakened.
Disclosure of Invention
Aiming at the performance requirement of the hydrogel dressing and the technical bottleneck or defect existing in the prior art, the invention designs a preparation method of the semi-interpenetrating network bioactive hydrogel. According to the method, two polymers of cellulose and polyglutamic acid are chemically crosslinked to form a rigid molecular skeleton, and then third polymer polylysine is inserted into the molecular skeleton to obtain the hydrogel. Through carrying out accurate design to parameters such as the kind, the dosage of three kinds of macromolecules, endow the hydrogel with excellent mechanical properties, thereby promoting the application effect and the clinical transformation potential thereof.
In a first aspect of the invention, a method for constructing semi-interpenetrating network bioactive hydrogel by secondary molding with simple process and industrial production prospect is provided, which comprises the following steps:
(1) Uniformly mixing a cellulose solution, polyglutamic acid and a crosslinking agent, and separating insoluble impurities in the mixture to obtain a mixed solution; solidifying the mixed solution to obtain a cellulose/polyglutamic acid copolymer;
(2) Removing residual impurities in the cellulose/polyglutamic acid copolymer to obtain covalent cross-linked hydrogel;
(3) Immersing the covalent cross-linked hydrogel in polylysine solution for treatment to obtain the semi-interpenetrating network bioactive hydrogel.
The polyglutamic acid and cellulose form chemical crosslinking, so that the crosslinking density of the hydrogel is increased, and the pore size is reduced; then polylysine is introduced, and a semi-interpenetrating network structure is formed by physical crosslinking of the amino group of the polylysine and the carboxyl group of the polyglutamic acid. The overmoulding further increases the cross-linking density of the hydrogel, so that the pore size of the hydrogel is further reduced, and the network structure is more dense.
After being mixed into covalent cross-linked hydrogel, the polylysine is used as a macromolecular compound with antibacterial property, and simultaneously, the amino group of the polylysine can be attracted with the carboxyl group of polyglutamic acid in the covalent cross-linked hydrogel to generate positive and negative charges and carry out secondary physical cross-linking, so that the double-network cross-linked hydrogel is formed, and the mechanical property of the hydrogel is enhanced.
According to the technical scheme, a secondary molding method is adopted, physical reversible adsorption crosslinking is carried out by utilizing the amino groups of the polylysine and the carboxyl groups of the polyglutamic acid, so that a semi-interpenetrating network structure is formed, the amino groups with antibacterial activity are not consumed by the structure, and meanwhile, the polylysine can be slowly released to a wound area by the semi-interpenetrating network structure, so that the hydrogel achieves better antibacterial performance.
Preferably, in the step (1), the cellulose solution is prepared by the following method in parts by weight: 7-8 parts of sodium hydroxide and 12-15 parts of urea are dissolved in 78-81 parts of ultrapure water, precooled to-13 to-12 ℃, and then 1-4 parts of cellulose is added and stirred until the cellulose is completely dissolved.
Preferably, in the step (1), the crosslinking agent is at least one of epichlorohydrin, ethylene glycol diglycidyl ether and glutaraldehyde.
Preferably, in the step (1), the content of cellulose in the mixed solution is 1-4%, the content of polyglutamic acid is 0.25-2%, and the content of the crosslinking agent is 2-5% by mass.
Preferably, in the step (1), the curing is performed at 10 to 40 ℃ for 4 to 24 hours.
Preferably, in the step (3), the polylysine solution is prepared by the following method in parts by weight: adding 0.05-5 parts of polylysine into 95-100 parts of ultrapure water, and stirring until the polylysine is completely dissolved.
Preferably, in the step (3), the ratio of the feed solution of the covalent cross-linked hydrogel to the polylysine solution is 1: 2-5, the unit of the feed liquid ratio is g/mL.
Preferably, in the step (3), the treatment is performed at 10 to 40 ℃ for 4 to 24 hours.
In a second aspect of the present invention, there is provided a semi-interpenetrating network bioactive hydrogel having good water absorption, antibacterial ability, and mechanical properties, the semi-interpenetrating network bioactive hydrogel being prepared by the method of the first aspect of the present invention.
In a third aspect of the invention there is provided the use of a semi-interpenetrating network bioactive hydrogel of the second aspect of the invention in the preparation of a hydrogel dressing.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. the invention provides a method for constructing semi-interpenetrating network bioactive hydrogel by secondary molding, which has the advantages of simple process, easily available processing equipment, high product addition and suitability for industrial production. The materials adopted in the production can be used for implantable medical devices, toxic chemical reagents are strictly limited in the preparation process, the safety of the product is ensured, and the strict requirements of clinical transformation of hydrogel dressing are met. The method does not consume amino with antibacterial activity, and gradually reduces the pore size of the hydrogel in a secondary forming mode, so that the reticular structure is denser.
2. The semi-interpenetrating network bioactive hydrogel provided by the invention has good water absorbability, biocompatibility and mechanical properties, can effectively inhibit the activities of escherichia coli and staphylococcus aureus, and can promote the skin repair process.
3. The invention provides application of semi-interpenetrating network bioactive hydrogel in preparing hydrogel dressing, which is suitable for various types of skin injuries, in particular to infectious skin diseases, diabetic ulcers and the like, and has good application prospect.
Drawings
FIG. 1a is a cross-linking process diagram of the hydrogel of example 1, ce representing cellulose, PGA representing polyglutamic acid, and PL representing polylysine; FIG. 1b shows the microscopic morphologies of the finished hydrogels of example 1 and comparative examples 1-2, CGLH, CH, CGH representing the finished hydrogels of example 1, comparative example 1, and comparative example 2, respectively; FIG. 1c is a photograph of the hydrogel of example 1;
FIG. 2a is a graph showing the compression mechanics of a CGLH, CH, CGH hydrogel; FIG. 2b is a graph of the elastic modulus corresponding thereto;
FIGS. 3a and 3c are photographs showing the inhibition of corresponding E.coli (E.coli) and Staphylococcus aureus (S.aureus), respectively, by different hydrogels; FIGS. 3b and 3d show colony counts corresponding to hydrogels; FIGS. 3e and 3f are the proliferation curves of E.coli (E.coli) and Staphylococcus aureus (S.aureus), respectively;
FIGS. 4a and 4b show the cell viability of different hydrogels after co-culturing with L929 cells, NIH-3T3 cells, respectively;
FIG. 5a is a photograph of wound healing in rats; FIG. 5b is a plot of the healing area of a rat wound; FIGS. 5 c-e are wound healing rates of rats on days 4, 8, 12, respectively; FIG. 5f is a hematoxylin & eosin (H & E) staining of rat wound cortex tissue sections; in the figure, b.c. represents a blank control group, and p.c. represents a control group.
Detailed Description
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
Example 1
The semi-interpenetrating network bioactive hydrogel is prepared by the following method:
(1) Weighing 3g of cellulose, tearing into small fragments, and putting into a refrigerator for refrigeration for standby; respectively weighing 7g of sodium hydroxide, 12g of urea and 81g of distilled water, stirring and dissolving in a beaker to obtain an alkali liquor, freezing the alkali liquor in a refrigerator, adding precooled cellulose fragments when the temperature of the alkali liquor reaches-12 ℃, and stirring until the pre-cooled cellulose fragments are completely dissolved to obtain a cellulose solution; taking 80g of the cellulose solution in a beaker, respectively adding 0.8g of polyglutamic acid and 3mL of epichlorohydrin into the cellulose solution, stirring the mixture until the mixture is uniformly mixed, centrifugally separating insoluble impurities in the mixture to obtain a mixed solution, pouring the mixed solution into a mould, curing the mixed solution at 25 ℃ for 12 hours, and forming a gel to obtain a cellulose/polyglutamic acid copolymer;
(2) Washing the cellulose/polyglutamic acid copolymer with water to remove impurities such as residual cross-linking agent, inorganic salt and the like, thereby obtaining covalent cross-linked hydrogel;
(3) 0.05g of polylysine is dissolved in 100mL of distilled water to obtain polylysine solution; according to the feed liquid ratio 1:2 (unit is g/mL) weighing the covalent cross-linked hydrogel with corresponding mass, then immersing the covalent cross-linked hydrogel in the polylysine solution completely, treating for 12h at 37 ℃, recovering the semi-interpenetrating network bioactive hydrogel after the treatment is completed, and marking the hydrogel as CGLH.
Example 2
(1) Weighing 1g of cellulose, tearing into small fragments, and putting into a refrigerator for refrigeration for standby; respectively weighing 7g of sodium hydroxide, 12g of urea and 81g of distilled water, stirring and dissolving in a beaker to obtain an alkali liquor, freezing the alkali liquor in a refrigerator, adding precooled cellulose fragments when the temperature of the alkali liquor reaches-12 ℃, and stirring until the pre-cooled cellulose fragments are completely dissolved to obtain a cellulose solution; taking 80g of the cellulose solution in a beaker, respectively adding 0.2g of polyglutamic acid and 1.6g of ethylene glycol diglycidyl ether into the cellulose solution, stirring the mixture until the mixture is uniformly mixed, centrifugally separating insoluble impurities in the mixture to obtain a mixed solution, pouring the mixed solution into a mould, curing the mixed solution at 10 ℃ for 24 hours, and forming a gel to obtain a cellulose/polyglutamic acid copolymer;
(2) Washing the cellulose/polyglutamic acid copolymer with water to remove impurities such as residual cross-linking agent, inorganic salt and the like, thereby obtaining covalent cross-linked hydrogel;
(3) 0.05g of polylysine is dissolved in 100mL of distilled water to obtain polylysine solution; according to the feed liquid ratio 1:2 (unit is g/mL) weighing covalent cross-linked hydrogel with corresponding mass, then immersing the covalent cross-linked hydrogel in the polylysine solution completely, treating for 24 hours at 10 ℃, and recovering after the treatment is completed to obtain the semi-interpenetrating network bioactive hydrogel.
Example 3
(1) Weighing 4g of cellulose, tearing into small fragments, and putting into a refrigerator for refrigeration for standby; respectively weighing 7g of sodium hydroxide, 12g of urea and 81g of distilled water, stirring and dissolving in a beaker to obtain an alkali liquor, freezing the alkali liquor in a refrigerator, adding precooled cellulose fragments when the temperature of the alkali liquor reaches-12 ℃, and stirring until the pre-cooled cellulose fragments are completely dissolved to obtain a cellulose solution; taking 80g of the cellulose solution in a beaker, respectively adding 1.6g of polyglutamic acid and 4g of glutaraldehyde into the cellulose solution, stirring the mixture until the mixture is uniformly mixed, centrifugally separating insoluble impurities in the mixture to obtain a mixed solution, pouring the mixed solution into a mould, curing the mixed solution at 40 ℃ for 4 hours, and forming a gel to obtain a cellulose/polyglutamic acid copolymer;
(2) Washing the cellulose/polyglutamic acid copolymer with water to remove impurities such as residual cross-linking agent, inorganic salt and the like, thereby obtaining covalent cross-linked hydrogel;
(3) Dissolving 5g of polylysine in 95mL of distilled water to obtain a polylysine solution; according to the feed liquid ratio 1:5 (unit is g/mL) weighing covalent cross-linked hydrogel with corresponding mass, then immersing the covalent cross-linked hydrogel in the polylysine solution completely, treating for 4 hours at 40 ℃, and recovering after the treatment is completed to obtain the semi-interpenetrating network bioactive hydrogel.
Comparative example 1
The cellulose hydrogel is prepared by the following method:
weighing 3g of cellulose, tearing into small fragments, and putting into a refrigerator for refrigeration for standby; respectively weighing 7g of sodium hydroxide, 12g of urea and 81g of distilled water, stirring and dissolving in a beaker to obtain an alkali liquor, freezing the alkali liquor in a refrigerator, adding precooled cellulose fragments when the temperature of the alkali liquor reaches-12 ℃, and stirring until the pre-cooled cellulose fragments are completely dissolved to obtain a cellulose solution; taking 80g of the cellulose solution in a beaker, adding 3mL of epichlorohydrin into the solution, stirring the solution until the solution is uniformly mixed, centrifugally separating insoluble impurities in the mixture to obtain a mixed solution, pouring the mixed solution into a mould, curing the mixed solution at 25 ℃ for 12 hours, and gelling to obtain cellulose hydrogel, wherein the hydrogel is marked as CH.
Comparative example 2
The cellulose/polyglutamic acid copolymer hydrogel is prepared by the following method:
(1) Weighing 3g of cellulose, tearing into small fragments, and putting into a refrigerator for refrigeration for standby; respectively weighing 7g of sodium hydroxide, 12g of urea and 81g of distilled water, stirring and dissolving in a beaker to obtain an alkali liquor, freezing the alkali liquor in a refrigerator, adding precooled cellulose fragments when the temperature of the alkali liquor reaches-12 ℃, and stirring until the pre-cooled cellulose fragments are completely dissolved to obtain a cellulose solution; taking 80g of the cellulose solution in a beaker, respectively adding 0.8g of polyglutamic acid and 3mL of epichlorohydrin into the cellulose solution, stirring the mixture until the mixture is uniformly mixed, centrifugally separating insoluble impurities in the mixture to obtain a mixed solution, pouring the mixed solution into a mould, curing the mixed solution at 25 ℃ for 12 hours, and forming a gel to obtain a cellulose/polyglutamic acid copolymer;
(2) And (3) washing the cellulose/polyglutamic acid copolymer with water to remove residual cross-linking agent, inorganic salt and other impurities, thereby obtaining cellulose/polyglutamic acid copolymer hydrogel, wherein the hydrogel is marked as CGH.
Test example 1
The preparation procedure and the physical pictures of example 1 were recorded, and the microscopic morphologies of the finished hydrogels (CGLH, CH, CGH) of example 1 and comparative examples 1 to 2 were observed by scanning electron microscopy. As can be seen from fig. 1, fig. 1a shows the cross-linking and gelling process of the hydrogel (CGLH) of example 1, and the semi-interpenetrating network bioactive hydrogel is constructed through a chemical and physical secondary cross-linking process; FIG. 1b shows the microscopic morphology of three hydrogels, with the same magnification, the diameters of the micropores of the hydrogels decrease with the introduction of polyglutamic acid and polylysine, and the network structure is denser, wherein the diameters of the micropores of CGLH are smaller than CH and CGH, and the optimal pore size is achieved, so that the optimization effect of secondary molding on the micropores is demonstrated; fig. 1c shows a physical picture of the hydrogel (CGLH) of example 1, which has excellent elasticity and bending properties and can be bent at any angle to the human joint without breaking.
Test example 2
The mechanical properties of the finished hydrogels (CGLH, CH, CGH) were tested. Three CGLH, CH, CGH sets of hydrogels were formed into standard cylindrical shapes with a diameter of 23mm and a height of 12mm, and compression strain was measured at a compression rate of 10mm/min, and 3 parallel experiments were performed for each sample, with the results being averaged. Meanwhile, the water absorption performance of the CGLH hydrogel is tested, and the result shows that the CGLH hydrogel has excellent water absorption (the water absorption rate of the CGLH hydrogel is 611.2 percent, the water absorption rate of the control group CH hydrogel is 382.7 percent, and the P is less than 0.05).
Fig. 2a and 2b show the compressive stress-strain curve and the compressive modulus histogram, respectively, of the hydrogels. As shown in the figure, compared with CH hydrogel, with the introduction of polyglutamic acid and polylysine, the mechanical property of CGLH hydrogel is greatly improved, and when the strain is 50%, the compression stress reaches 65.7kPa (19.95 KPa, P <0.05 for control group CH hydrogel); the elastic modulus is 0.44MPa, and the mechanical property of the CGLH hydrogel is 3-4 times that of the CH hydrogel.
Test example 3
Freeze-drying the finished hydrogel (CGLH, CH, CGH), grinding into powder, and placing in a test tube; to each tube was added 2mL of sterilized LB medium and the mixture was shaken at a constant temperature of 37℃for 4 hours at 200 rpm. The test strains are escherichia coli (E.coli) and staphylococcus aureus (S.aureus), respectively diluting the LB culture medium with the bacteria step by step, then coating the LB solid culture plate with the bacteria, and culturing the bacteria in an inversion way at 37 ℃ overnight. And taking a colony photo by adopting a digital camera, and obtaining the antibacterial rate of the hydrogel through statistical analysis.
FIGS. 3a and 3c show photographs of hydrogels inhibiting growth of corresponding colonies; FIGS. 3b and 3d show colony counts of hydrogels; FIGS. 3e and 3f show the proliferation curves of bacteria. As shown in the figure, the hydrogel has excellent inhibition effect on escherichia coli (E.coli) and staphylococcus aureus S.aureus), can effectively prevent bacteria propagation in a wound area, and creates a good healing environment for the wound.
Test example 4
L929 cells and NIH-3T3 cells were inoculated onto 24-well tissue culture plates, respectively, and co-cultured with hydrogel (CGLH, CH, CGH) for 48 hours. The blank (con.) group was not treated. At regular intervals, 10% MTT (3- (4, 5-dimethyl-2-thiazole) -2, 5-diphenyl tetrazolium bromide thiazole blue) reagent was added to each sample, and incubated for 4 hours. Thereafter, the medium in each sample was removed and replaced with 1mL DMSO solution. After stirring in the dark for 10min, the absorbance of the supernatant at 490nm was measured with a microplate reader.
FIGS. 4a and 4b show the cell viability of hydrogels after co-culturing with L929 cells and NIH-3T3 cells, respectively. For acceptable biological materials, the cell viability should not be less than 80%. After the CGLH hydrogel is co-cultured with cells, the activity of the L929 and NIH-3T3 cells is not affected basically, the relative proliferation rate of the L929 cells reaches 92.4%, and the relative proliferation rate of the NIH-3T3 cells reaches 93.2%. The CGLH hydrogel has good biocompatibility.
Test example 5
The CGLH and CH hydrogels were cut into wafers with a diameter of 20mm, and the skin lesion healing effect of the hydrogels was verified by means of rat full-cortex excision. Rats were purchased from the disease prevention and control center in Hubei province, and after isoflurane anesthesia, the full-cortical defect wound surface with the diameter of 20mm was excised at the back of the rat (operation)The process and feeding were performed according to the security assessment center (Fu) No. 202220085). The blank control group (b.c.) was applied without any material, CGLH, CH were used as the experimental group, the experimental group was applied to the wound surface, and the control group (p.c.) was a commercially available hydrocolloid material (us SILVERCEL TM Silver-containing dressing), and conventional rearing after operation. Taking photographs of the wound at intervals, performing image processing and statistics to obtain a full-cortex healing state, slicing a tissue sample by paraffin, and performing hematoxylin treatment&Eosin (H)&E) After staining, observation was performed. And calculating the wound healing area by using imageJ software, and counting to obtain the wound healing rate.
Fig. 5a shows a photograph of wound healing in rats; fig. 5b shows the healing area of a rat wound; figures 5 c-e show wound healing rates of rats on days 4, 8, 12, respectively; FIG. 5f shows H of a rat wound cortex tissue section&E staining. As shown, the CGLH group healed most quickly from a histological point of view. The local magnification can observe the new granulation tissue, and the four groups form complete epithelial tissues. Group p.c. epithelial cells begin to keratinize. In terms of immune response, the b.c. and CH groups have massive inflammatory cell infiltration, in part due to the proliferation of e. The p.c. and CGLH groups had less inflammatory cell infiltration. The bacteriostatic activity of the P.C. group mainly comes from Ag + The bacteriostatic activity of the CGLH group is derived from epsilon-polylysine. The biological safety of epsilon-polylysine is obviously higher than Ag + . Both the p.c. group and the CGLH group see a large number of blood vessels. The blood vessels are capable of transporting oxygen and nutrients and promoting regeneration of host cells. On the other hand, the extracellular matrix (ECM) of the p.c. group and CGLH group is also produced in large quantities and initially reconstituted. From H&The E result shows that the wound healing effect of the CGLH group is almost equivalent to that of the P.C. group.
The foregoing describes in detail preferred embodiments of the present invention. It should be understood that numerous modifications and variations can be made in accordance with the concepts of the invention by one of ordinary skill in the art without undue burden. Therefore, all technical solutions which can be obtained by logic analysis, reasoning or limited experiments based on the prior art by the person skilled in the art according to the inventive concept shall be within the scope of protection defined by the claims.
Claims (10)
1. A method for constructing semi-interpenetrating network bioactive hydrogel by secondary molding, comprising the following steps:
(1) Uniformly mixing a cellulose solution, polyglutamic acid and a crosslinking agent, and separating insoluble impurities in the mixture to obtain a mixed solution; solidifying the mixed solution to obtain a cellulose/polyglutamic acid copolymer;
(2) Removing residual impurities in the cellulose/polyglutamic acid copolymer to obtain covalent cross-linked hydrogel;
(3) Immersing the covalent cross-linked hydrogel in polylysine solution for treatment to obtain the semi-interpenetrating network bioactive hydrogel.
2. The method according to claim 1, wherein in the step (1), the cellulose solution is prepared by the following method in parts by weight: 7-8 parts of sodium hydroxide and 12-15 parts of urea are dissolved in 78-81 parts of ultrapure water, precooled to-13 to-12 ℃, and then 1-4 parts of cellulose is added and stirred until the cellulose is completely dissolved.
3. The method according to claim 1, characterized in that: in the step (1), the cross-linking agent is at least one of epichlorohydrin, ethylene glycol diglycidyl ether and glutaraldehyde.
4. The method according to claim 1, characterized in that: in the step (1), the content of cellulose in the mixed solution is 1-4%, the content of polyglutamic acid is 0.25-2%, and the content of the crosslinking agent is 2-5% by mass percent.
5. The method according to claim 1, characterized in that: in the step (1), the curing is carried out at the temperature of 10-40 ℃ and the curing time is 4-24 hours.
6. The method according to claim 1, wherein in the step (3), the polylysine solution is prepared by the following method in parts by weight: adding 0.05-5 parts of polylysine into 95-100 parts of ultrapure water, and stirring until the polylysine is completely dissolved.
7. The method according to claim 1, characterized in that: in the step (3), the feed liquid ratio of the covalent crosslinking hydrogel to the polylysine solution is 1: 2-5, the unit of the feed liquid ratio is g/mL.
8. The method according to claim 1, characterized in that: in the step (3), the treatment is carried out at the temperature of 10-40 ℃ for 4-24 hours.
9. A semi-interpenetrating network bioactive hydrogel, characterized by: the method according to any one of claims 1 to 8.
10. Use of the semi-interpenetrating network bioactive hydrogel of claim 9 in the preparation of a hydrogel dressing.
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