CN114848893A - Silk fibroin-combined decellularized small intestine submucosa extracellular matrix hydrogel for pigs, and preparation method and application thereof - Google Patents
Silk fibroin-combined decellularized small intestine submucosa extracellular matrix hydrogel for pigs, and preparation method and application thereof Download PDFInfo
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- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
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- A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
- A61L26/0028—Polypeptides; Proteins; Degradation products thereof
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- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
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Abstract
The invention discloses a silk fibroin-combined porcine decellularized small intestine submucosa extracellular matrix hydrogel as well as a preparation method and application thereof, belonging to the technical field of hydrogels. A preparation method of a silk fibroin-combined porcine decellularized small intestine submucosa extracellular matrix hydrogel comprises the following steps: digesting the decellularized small intestine submucosa, salting out, freeze-drying, preparing into powder, dissolving, mixing with the silk fibroin solution, and performing ultrasonic treatment to obtain the composition. The extracellular matrix hydrogel can be used as a skin injury repair dressing and a biocompatible cell scaffold. After the silk fibroin is combined, the wound healing speed and the skin healing degree are both obviously improved.
Description
Technical Field
The invention relates to a silk fibroin-combined porcine decellularized small intestine submucosa extracellular matrix hydrogel as well as a preparation method and application thereof, belonging to the technical field of hydrogels.
Background
Extracellular matrix hydrogel derived from decellularized tissue has been widely used in the field of tissue engineering as a scaffold material with good biocompatibility. The small intestine submucosa subjected to decellularization treatment mainly contains type I and type III collagens, rich fibronectin, growth factors and the like, and is an ideal cell scaffold material for protecting wound surfaces and promoting tissue healing. The silk fibroin is a natural biological material extracted from silkworm silk and has good biocompatibility. At present, clinically, the problems of skin source deficiency, immunological rejection and the like still exist in the process of skin transplantation after skin injury in wound repair treatment, so that the search for a biological material which has good biocompatibility and convenient material acquisition and can be prepared in batches becomes very important.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a silk fibroin-combined porcine decellularized small intestine submucosa extracellular matrix hydrogel as well as a preparation method and application thereof.
The invention provides a preparation method of silk fibroin-combined porcine decellularized small intestine submucosa extracellular matrix hydrogel, which comprises the following steps: digesting the decellularized small intestine submucosa, salting out, freeze-drying, preparing into powder, dissolving, mixing with the silk fibroin solution, and performing ultrasonic treatment to obtain the composition.
Further, in the above technical scheme, the preparation method comprises the following steps:
(1) digesting the powder of the lower mucous membrane layer of the decellularized small intestine of the pig by using digestive juice, centrifuging, taking supernatant, and adjusting the pH to 7-7.5; adding salt to separate out, centrifuging for several times, retaining precipitate, and freeze drying;
(2) dissolving dried starch powder in 50-100mg/ml 0.1-0.2% (v/v) acetic acid, and regulating osmotic pressure to physiological level with 10 xMEM;
(3) 3-6% (w/v) of silk fibroin solution is subjected to ultrasonic treatment, and the solution obtained in the step (2) is added into the silk fibroin solution after ultrasonic treatment according to the volume ratio of 1:1-1: 1.5;
(4) and (4) ultrasonically treating the mixed solution, inoculating the mixed solution into a pore plate, and incubating to obtain the compound.
Further, in the above technical scheme, the decellularized small intestine submucosa powder for pigs is prepared by degreasing, decellularizing, digesting with pepsin, performing salting-out centrifugation for multiple times, performing vacuum freeze drying, crushing and grinding on the small intestine submucosa.
Further, in the technical scheme, the degreasing is that the submucosa of the small intestine of the pig is kept standing in a solution with the volume ratio of methanol to chloroform of 1:1-1:1.5 in a dark place.
Further, in the above technical scheme, the decellularization is to soak the defatted porcine small intestine submucosa in a solution of 0.04-0.06% (w/v) trypsin/0.04-0.06% (w/v) disodium edetate prepared by deionized water, and shake the solution in 0.4-0.6% (w/v) SDS and physiological saline for 4-5 hours.
Further, in the technical scheme, the disinfection and cleaning are that the small intestine submucosa of the decellularized pig is soaked in 0.1-0.2% (w/v) peroxyacetic acid and 15-25% (v/v) ethanol; and (4) thoroughly washing with deionized water to remove residual reagent, and replacing the deionized water until no foam is generated by shaking.
Further, in the technical scheme, the digestive juice in the step (1) is pepsin digestive juice, the mass ratio of the porcine decellularized small intestine submucosa to the pepsin digestive enzyme is 10:1, then 3% acetic acid is added according to 10mg/ml, and after 48-72 hours, the porcine decellularized small intestine submucosa is semitransparent and centrifuged without floccules.
The invention also provides the silk fibroin-combined porcine decellularized small intestine submucosa extracellular matrix hydrogel prepared by the preparation method.
The invention also provides application of the pig decellularized small intestine submucosa extracellular matrix hydrogel combined with the silk fibroin in preparation of the dressing for repairing skin injury.
The invention also provides application of the cell-free small intestine submucosa extracellular matrix hydrogel combined with the silk fibroin in preparation of a biocompatible cell scaffold.
Advantageous effects of the invention
The silk fibroin-combined porcine decellularized small intestine submucosa extracellular matrix hydrogel is simple in preparation method, low in cost, capable of realizing batch preparation, good in biocompatibility and capable of being used as a skin injury repair dressing and a biocompatible cell scaffold. After the silk fibroin is combined, the wound healing speed and the skin healing degree are both obviously improved.
Drawings
FIG. 1 shows the general appearance of the skin defect recovery of the groups of model mice in application example 1.
FIG. 2 is a photograph of Hematoxylin and Eosin (H & E) staining (indicated at 200 μm) after repair of skin defects in mice using the groups of models in example 1.
Fig. 3 is a photograph of CK15 Immunohistochemical (IHC) staining after repair of skin defects of each group of model mice in application example 1 (legend 50 μm).
Detailed Description
The following non-limiting examples will allow one of ordinary skill in the art to more fully understand the present invention, but are not intended to limit the invention in any way.
Example 1 preparation of decellularized porcine small intestine submucosa extracellular matrix hydrogel
1. Preparation of decellularized small intestine submucosa tissue
The small intestine of a healthy adult pig is selected, and the part with uniform tube cavity thickness, no damage to the tube wall and no lymph node is selected. The small intestine is cleaned, and serosa and muscular layer tissues are removed. Turning over the small intestine to make the mucosa face outwards, removing the mucosa layer of the small intestine until the submucosa is exposed, cleaning the submucosa of the small intestine, and completely removing the residual tissues on the submucosa of the small intestine. The pretreated SIS (small intestinal submucosa) is soaked in a solution of methanol and chloroform mixed according to the volume ratio of 1:1 and kept in the dark at room temperature for 12 hours for degreasing. 0.05% trypsin and 0.05% EDTA-Na2 disodium EDTA solution in deionized water, and the cells were removed by shaking in a shaker for 12 hours, 0.5% SDS and 0.9% NaCl solution for 4 hours. 0.1% peracetic acid and 20% ethanol for 30 minutes, rinsed thoroughly with deionized water, to remove residual reagents, and shaken on a shaker for 48 hours. The SIS after cell removal is placed in a refrigerator at the temperature of 80 ℃ below zero for pre-freezing, and then is vacuumized and freeze-dried for 24 to 48 hours.
2. Preparation of hydrogels
Pulverizing, lyophilizing, mixing at a ratio of 10mg SIS/1mg pepsin/ml 3% acetic acid, and digesting with magnetic stirrer at room temperature for 48 hr, not more than 72 hr. Digesting to obtain SIS without floccule and semitransparent. Digestion was stopped by adjusting the pH to 7.5 with 2M NaOH, 4 ℃, 9000rpm, centrifugation for 15 minutes and supernatant was taken. 80mg/ml NaCl was added and centrifuged (4 ℃, 9000rpm, 15 minutes), the supernatant was discarded, and the precipitate was dissolved by adding ultrapure water, and salting out was performed again, and this step was repeated three times. And (4) precooling the precipitate in a refrigerator at-80 ℃, and then vacuumizing and freeze-drying. Redissolving at 50-100mg lyophilized pellet/ml 0.1% acetic acid, adjusting the osmotic pressure to physiological levels (volume of redissolution/9) with 10 xDMM, and adjusting the pH to 7.5 (volume of DMEM/20) with 2M NaOH. Standing the solution at 4 deg.C overnight, inoculating 400 μ l into 24-well plate, placing in 37 deg.C incubator for 1 hr, and gelatinizing.
Example 2 preparation of Silk Fibroin (SF) -binding decellularized porcine small intestine submucosa extracellular matrix hydrogel
1. Preparation of decellularized small intestine submucosa tissue
The small intestine of a healthy adult pig is selected, and the part with uniform tube cavity thickness, no damage to the tube wall and no lymph node is selected. The small intestine is cleaned, and serosa and muscular layer tissues are removed. Turning over the small intestine to make the mucosa face outwards, removing the mucosa layer of the small intestine until the submucosa is exposed, cleaning the submucosa of the small intestine, and completely removing the residual tissues on the submucosa of the small intestine. Soaking the pretreated SIS in a solution of methanol and chloroform in a volume ratio of 1:1, standing at room temperature in a dark place for 12 hours, and degreasing. 0.05% trypsin and 0.05% EDTA-Na2 in deionized water, and the mixture was placed in a shaker for 12 hours, 0.5% SDS and 0.9% NaCl for 4 hours and shaken continuously in a shaker. 0.1% peracetic acid and 20% ethanol for 30 minutes, rinsed thoroughly with deionized water, to remove residual reagents, and shaken on a shaker for 48 hours. The SIS after cell removal is placed in a refrigerator at the temperature of 80 ℃ below zero for pre-freezing, and then is vacuumized and freeze-dried for 24 to 48 hours.
2. Preparation of hydrogels
Crushing and freeze-drying the SIS, adding pepsin according to the mass ratio of the SIS to the pepsin of 10:1, adding 3% acetic acid according to the mass ratio of 10mg/ml, and digesting and dissolving for 48 hours at room temperature by using a magnetic stirrer, wherein the time is not more than 72 hours. Digesting until the SIS is flocculent-free and translucent. Digestion was stopped by adjusting the pH to 7.5 with 2M NaOH, 4 ℃, 9000rpm, centrifugation for 15 minutes and supernatant was taken. NaCl was added to the supernatant at 80mg/ml, the mixture was centrifuged at 9000rpm at 4 ℃ for 15 minutes, the supernatant was discarded, and ultrapure water was added to dissolve the precipitate, followed by salting out again, and this step was repeated three times. And (4) precooling the precipitate in a refrigerator at-80 ℃, and then vacuumizing and freeze-drying. The lyophilized precipitate was pulverized, dissolved in 50-100mg/ml 0.1% acetic acid, and the permeate was adjusted with 10 xDMM (1/9), pH adjusted with 2M NaOH (DMEM volume/20). Standing the solution at 4 deg.C overnight, ultrasonically treating SF solution, and 3-6% SF solution for 10s (AMP 20%, pulse10), and adding the treated SIS solution into the SF solution at a ratio of 1:1. The mixed solution is subjected to ultrasonic treatment for 10s again, 400 mu l of the mixed solution is inoculated to a 24-well plate and is placed in an incubator at 37 ℃ for 1 hour, and then the gel is formed.
Application example 1
This application example serves to illustrate the effect of the gels of the present invention on wound healing. Mice of 4-6 weeks old are used as experimental animals, 10% chloral hydrate is used for abdominal cavity anesthesia, and skin preparation and disinfection are carried out. A circular incision of 1cm in diameter was made in the dorsal skin and a full-thickness skin excision was performed. The experimental group 1 applied the porcine decellularized small intestine submucosa extracellular matrix hydrogel to the wound site, and the experimental group 2 applied the porcine decellularized small intestine submucosa extracellular matrix hydrogel mixed with SF to the wound site. Gauze was used for the control group. All mice were covered with 3M Tegaderm transparent dressing over the whole wound site, bandaged and fixed.
The results are shown in fig. 1, and the wound healing rate of the experimental group 1 and the experimental group 2 is obviously faster than that of the control group in the healing process. H & E staining was performed on the tissues of the healed wound sites (FIG. 2), and the results showed that the experimental group showed epidermis formation, better restoration of the dermal layer structure and more number of regenerated hair follicles at 7 and 14 days compared with the control group, wherein the number of hair follicles of the decellularized small intestine submucosa extracellular matrix hydrogel of the pigs after mixing with SF was slightly more than that of the group without addition of SF. IHC staining (CK15) (FIG. 3) showed that the hair follicle differentiation degree was better in the experimental group, and the skin lesion recovery of mice with SF was closer to the normal skin tissue morphology at 14 days, and it was observed that the skin healing degree and speed in the experimental group were higher than those of the control group and the SF-added group.
Claims (10)
1. A preparation method of silk fibroin-combined porcine decellularized small intestine submucosa extracellular matrix hydrogel is characterized by comprising the following steps: the method comprises the following steps: digesting the decellularized small intestine submucosa, salting out, freeze-drying, preparing into powder, dissolving, mixing with the silk fibroin solution, and performing ultrasonic treatment to obtain the composition.
2. The method of claim 1, wherein: the method comprises the following steps:
(1) digesting the powder of the lower mucous membrane layer of the decellularized small intestine of the pig by using digestive juice, centrifuging, taking supernatant, and adjusting the pH to 7-7.5; adding salt to separate out, centrifuging for several times, retaining precipitate, and freeze drying;
(2) dissolving dried starch powder in 50-100mg/ml 0.1-0.2% (v/v) acetic acid, and regulating osmotic pressure with DMEM to physiological level;
(3) 3-6% (w/v) of silk fibroin solution is subjected to ultrasonic treatment, and the solution obtained in the step (2) is added into the silk fibroin solution after ultrasonic treatment according to the volume ratio of 1:1-1: 1.5;
(4) and (4) performing ultrasonic treatment on the mixed solution, inoculating the mixed solution into a pore plate, and incubating to obtain the compound.
3. The method of claim 2, wherein: the porcine decellularized small intestine submucosa powder is prepared by carrying out degreasing, decellularization, pepsin digestion, salting out and centrifugation for multiple times, vacuum freeze drying, crushing and grinding on the porcine small intestine submucosa.
4. The production method according to claim 3, characterized in that: the degreasing is that the submucosa of the small intestine of the pig is kept standing in a dark place in a solution of methanol and chloroform with the volume ratio of 1:1-1: 1.5.
5. The production method according to claim 3, characterized in that: the decellularization is to soak the degreased small intestine submucosa of the pig in a solution of 0.04-0.06% (w/v) trypsin/0.04-0.06% (w/v) disodium ethylene diamine tetraacetate prepared by deionized water, and then shake the solution in 0.4-0.6% (w/v) SDS and physiological saline for 4-5 hours.
6. The production method according to claim 3, characterized in that: the disinfection and cleaning are that the porcine small intestine submucosa after cell removal is soaked in 0.1-0.2% (w/v) peroxyacetic acid and 15-25% (v/v) ethanol; and (4) thoroughly washing with deionized water to remove residual reagent, and replacing the deionized water until no foam is generated by shaking.
7. The production method according to claim 3, characterized in that: the digestive juice in the step (1) is pepsin digestive juice, the mass ratio of the porcine acellular small intestine submucosa to the pepsin digestive enzyme is 9:1-11:1, 2.5-3.5% (v/v) acetic acid is added according to 9.5-10.5mg/ml, and after 48-72h, the porcine acellular small intestine submucosa is semitransparent and centrifuged without floccules.
8. The silk fibroin-bound porcine decellularized small intestine submucosa extracellular matrix hydrogel prepared by the preparation method of any one of claims 1-7.
9. The use of the silk fibroin-binding porcine decellularized small intestine submucosa extracellular matrix hydrogel of claim 8 in the preparation of a dressing for repairing skin injury.
10. The use of the silk fibroin-conjugated porcine decellularized small intestine submucosa extracellular matrix hydrogel of claim 8 in the preparation of a biocompatible cell scaffold.
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