CN114848893B - Pig decellularized small intestine submucosa extracellular matrix hydrogel combined with silk fibroin and preparation method and application thereof - Google Patents
Pig decellularized small intestine submucosa extracellular matrix hydrogel combined with silk fibroin and preparation method and application thereof Download PDFInfo
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- 102000010834 Extracellular Matrix Proteins Human genes 0.000 title claims abstract description 22
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- 239000002244 precipitate Substances 0.000 claims description 10
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- RNFNDJAIBTYOQL-UHFFFAOYSA-N chloral hydrate Chemical compound OC(O)C(Cl)(Cl)Cl RNFNDJAIBTYOQL-UHFFFAOYSA-N 0.000 description 1
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- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
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- 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
- A61L26/0028—Polypeptides; Proteins; Degradation products thereof
- A61L26/0047—Specific proteins or polypeptides not covered by groups A61L26/0033 - A61L26/0042
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- 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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Abstract
The invention discloses a pig decellularized small intestine submucosa extracellular matrix hydrogel combined with silk fibroin and a preparation method and application thereof, and belongs to the technical field of hydrogels. A method for preparing a pig decellularized small intestine submucosa extracellular matrix hydrogel combined with silk fibroin, which comprises the following steps: digestion of decellularized intestinal submucosa, salting out, freeze drying, making into powder, dissolving, mixing with silk fibroin solution, and ultrasound. The extracellular matrix hydrogel can be used as a skin injury repair dressing and a biocompatible cell scaffold. After silk fibroin is combined, the wound healing speed and the skin healing degree are obviously improved.
Description
Technical Field
The invention relates to a pig decellularized small intestine submucosa extracellular matrix hydrogel combined with silk fibroin and a preparation method and application thereof, and belongs to the technical field of hydrogels.
Background
Extracellular matrix hydrogels derived from decellularized tissue have been widely used in the field of tissue engineering as a scaffold material with good biocompatibility. The small intestine submucosa after 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, in the clinic, the problems of insufficient skin source, immune rejection and the like still exist in the transplantation after the skin injury in the wound repair treatment, so that the searching of a biological material which has good biocompatibility, convenient material acquisition and batch preparation becomes very important.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a pig decellularized small intestine submucosa extracellular matrix hydrogel combined with silk fibroin, and a preparation method and application thereof.
The invention provides a preparation method of pig decellularized small intestine submucosa extracellular matrix hydrogel combined with silk fibroin, which comprises the following steps: digestion of decellularized intestinal submucosa, salting out, freeze drying, making into powder, dissolving, mixing with silk fibroin solution, and ultrasound.
Further, in the above technical solution, the preparation method includes the following steps:
(1) Digesting the pig decellularized small intestine submucosa powder with digestive juice, centrifuging, collecting supernatant, and adjusting pH to 7-7.5; adding salt, separating out, centrifuging for several times, retaining precipitate, and freeze drying;
(2) Dissolving the dried precipitate powder with 0.1-0.2% (v/v) acetic acid at a concentration of 50-100mg/ml, and regulating osmotic pressure to physiological level with 10 xDPMEM;
(3) 3-6% (w/v) of silk fibroin solution is subjected to ultrasound, and the solution obtained in the step (2) is added into the ultrasound silk fibroin solution according to the volume ratio of 1:1-1:1.5;
(4) And (3) carrying out ultrasonic treatment on the mixed solution, inoculating the mixed solution into a pore plate, and incubating to obtain the composite material.
Furthermore, in the technical scheme, the pig decellularized small intestine submucosa powder is prepared by degreasing, decellularizing, pepsin digestion, repeated salting-out centrifugation, vacuum freeze drying, crushing and grinding the pig small intestine submucosa.
Further, in the technical scheme, degreasing is that the submucosa of the small intestine of the pig is kept stand in a dark place in a solution with the volume ratio of methanol to chloroform of 1:1-1:1.5.
Further, in the above technical scheme, the decellularization is to soak the defatted pig small intestine submucosa in a solution of 0.04-0.06% (w/v) trypsin/0.04-0.06% (w/v) disodium ethylenediamine tetraacetate prepared by deionized water, and shake in 0.4-0.6% (w/v) SDS and physiological saline for 4-5 hours.
Further, in the technical scheme, the sterilization and the cleaning are that the decellularized pig small intestine submucosa is soaked in 0.1-0.2% (w/v) peracetic acid and 15-25% (v/v) ethanol; thoroughly rinsing with deionized water, removing residual reagent, and replacing deionized water until shaking is free of foam.
Further, in the above technical scheme, the digestive juice in the step (1) is pepsin digestive juice, the mass ratio of the pig decellularized small intestine submucosa to the gastric protein digestive enzyme is 10:1, 3% acetic acid is added according to 10mg/ml, and after 48-72 hours, the pig decellularized small intestine submucosa is semitransparent and centrifugal.
The invention also provides the pig decellularized small intestine submucosa extracellular matrix hydrogel combined with the silk fibroin, which is prepared by the preparation method.
The invention also provides application of the pig decellularized small intestine submucosa extracellular matrix hydrogel combined with silk fibroin in preparing a skin injury repair dressing.
The invention also provides application of the pig decellularized small intestine submucosa extracellular matrix hydrogel combined with silk fibroin in preparation of a biocompatible cell scaffold.
Advantageous effects of the invention
The preparation method of the pig decellularized small intestine submucosa extracellular matrix hydrogel combined with the silk fibroin is simple, 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 silk fibroin is combined, the wound healing speed and the skin healing degree are obviously improved.
Drawings
FIG. 1 is a general view of recovery of skin defects in each group of model mice in application example 1.
FIG. 2 is a photograph (200 μm in legend) of Hematoxylin and Eosin (H & E) staining after repair of skin defects in each group of model mice in application example 1.
FIG. 3 is a photograph (legend 50 μm) of CK15 immunohistochemistry (Immunohistochemical staining, IHC) staining after repair of skin defects in each group of model mice in application example 1.
Detailed Description
The following non-limiting examples will enable those of ordinary skill in the art to more fully understand the invention and are not intended to limit the invention in any way.
EXAMPLE 1 preparation of extracellular matrix hydrogels of decellularized porcine small intestine submucosa
1. Preparation of decellularized small intestine submucosa tissue
Small intestines of healthy adult pigs are taken, and the parts with uniform thickness of the lumen, no damage to the tube wall and no lymph nodes are selected. The small intestine was cleaned, and serosa and musculature were cleared. The small intestine is turned over to enable the mucous membrane surface to face outwards, the mucous membrane layer of the small intestine is removed until the mucous membrane subsurface layer is exposed, the mucous membrane subsurface layer of the small intestine is cleaned, and residual tissues on the mucous membrane subsurface layer of the small intestine are completely removed. The pretreated SIS (small intestine submucosa) is soaked in a solution of methanol and chloroform mixed according to the volume ratio of 1:1, and the mixture is kept away from light for 12 hours for degreasing. Decellularization in 0.05% trypsin (trypsin) and 0.05% EDTA-Na2 (disodium ethylenediamine tetraacetate) solution prepared by deionized water, placing in a shaking table for continuous shaking for 12 hours, decellularization in 0.5% SDS and 0.9% NaCl solution, and continuous shaking for 4 hours. Sterilizing in 0.1% peracetic acid and 20% ethanol for 30min, thoroughly washing with deionized water, removing residual reagent, and shaking the shaker for 48 hr. The acellular SIS is placed in a refrigerator at the temperature of minus 80 ℃ for pre-freezing, and then is vacuumized, frozen and dried for 24 to 48 hours.
2. Preparation of hydrogels
After lyophilization, the powder was mixed in a ratio of 10mg SIS/1mg pepsin/ml 3% acetic acid, and dissolved by magnetic stirrer at room temperature for 48 hours, not more than 72 hours. Digestion to SIS without floc, semitransparent. Digestion was stopped by adjusting the pH to 7.5 with 2M NaOH, centrifuging at 4℃and 9000rpm for 15 minutes and taking the supernatant. 80mg/ml NaCl was added and centrifuged (4 ℃,9000rpm,15 min), the supernatant was discarded, the precipitate was dissolved by adding ultrapure water, and the procedure was repeated three times. Pre-cooling the precipitate in a refrigerator at-80 deg.c, and vacuum freeze drying. Redissolving at a rate of 50-100mg of the lyophilized and pulverized precipitate/ml of 0.1% acetic acid, adjusting osmotic pressure to physiological level (redissolved solution volume/9) with 10 xmem, and adjusting pH to 7.5 (DMEM volume/20) with 2M NaOH. Standing the solution at 4deg.C overnight, inoculating 400 μl into 24-well plate, and placing into a 37-degree incubator for 1 hr to get gel.
Example 2 preparation of decellularized porcine small intestine submucosa extracellular matrix hydrogel in combination with silk fibroin (Silk fibroin, SF)
1. Preparation of decellularized small intestine submucosa tissue
Small intestines of healthy adult pigs are taken, and the parts with uniform thickness of the lumen, no damage to the tube wall and no lymph nodes are selected. The small intestine was cleaned, and serosa and musculature were cleared. The small intestine is turned over to enable the mucous membrane surface to face outwards, the mucous membrane layer of the small intestine is removed until the mucous membrane subsurface layer is exposed, the mucous membrane subsurface layer of the small intestine is cleaned, and residual tissues on the mucous membrane subsurface layer of the small intestine are completely removed. The pretreated SIS is soaked in a solution of methanol and chloroform mixed according to the volume ratio of 1:1, and the solution is kept away from light for 12 hours for degreasing. Decellularization in 0.05% trypsin and 0.05% EDTA-Na2 solution in deionized water was carried out by continuously shaking on a shaker for 12 hours, decellularization in 0.5% SDS and 0.9% NaCl solution was carried out by continuously shaking on a shaker for 4 hours. Sterilizing in 0.1% peracetic acid and 20% ethanol for 30min, thoroughly washing with deionized water, removing residual reagent, and shaking the shaker for 48 hr. The acellular SIS is placed in a refrigerator at the temperature of minus 80 ℃ for pre-freezing, and then is vacuumized, frozen and dried for 24 to 48 hours.
2. Preparation of hydrogels
Crushing and freeze-drying SIS, adding pepsin according to the mass ratio of SIS to pepsin of 10:1, adding 3% acetic acid according to 10mg/ml, and performing digestion and dissolution for 48 hours at room temperature, which is not more than 72 hours. Digestion to SIS without floc, semitransparent. Digestion was stopped by adjusting the pH to 7.5 with 2M NaOH, centrifuging at 4℃and 9000rpm for 15 minutes and taking the supernatant. NaCl is added into the supernatant at 80mg/ml, the supernatant is removed by centrifugation at 9000rpm for 15 minutes at 4 ℃, ultrapure water is added to dissolve the precipitate, the salting-out is performed again, and the step is repeated three times. Pre-cooling the precipitate in a refrigerator at-80 deg.c, and vacuum freeze drying. The lyophilized precipitate was crushed, dissolved in 0.1% acetic acid at 50-100mg/ml, permeate (1/9) was adjusted with 10xDMEM, and pH (DMEM volume/20) was adjusted with 2M NaOH. Standing the solution at 4 ℃ overnight, ultrasonically treating the SF solution with 3-6% of the SF solution for 10s (AMP 20%, pulse 10), and adding the ultrasonic SF solution into the treated SIS solution according to the ratio of 1:1. The mixed solution was sonicated again for 10s, 400. Mu.l was inoculated in 24 well plates and placed in a 37℃incubator for 1 hour to gel.
Application example 1
This application example is intended to illustrate the effect of the gel of the present invention on wound healing. Taking 4-6 week old mice as experimental animals, and 10% chloral hydrate is used for peritoneal anesthesia and skin preparation and disinfection. The back skin was cut in a circular shape with a diameter of 1cm, and the whole skin was excised. Experiment group 1, pig decellularized small intestine submucosa extracellular matrix hydrogel is applied to a wound site, and experiment group 2, pig decellularized small intestine submucosa extracellular matrix hydrogel mixed with SF is applied to the wound site. Gauze was used for the control group. All mice were covered with a 3M tergaderm transparent dressing over the whole wound site and bandaged.
The results are shown in fig. 1, and the experimental group 1 and the experimental group 2 were observed, and the wound healing rate was significantly faster than that of the control group in the healing process. H & E staining of the wound site tissue (fig. 2) showed that the experimental group showed better recovery of dermal layer structure and a greater number of regenerated hair follicles on days 7 and 14 than the control group, wherein the number of pig decellularized small intestine submucosa extracellular matrix hydrogel hair follicles after SF mixing was slightly greater than that of the group without SF addition. IHC staining (CK 15) (fig. 3) showed better differentiation of hair follicles in the experimental group, and at 14 days, mice with SF added recovered more nearly to normal skin tissue morphology, and skin healing was observed to be higher and faster than in the control group and for the SF added group.
Claims (4)
1. A method for preparing a silk fibroin-combined pig decellularized small intestine submucosa extracellular matrix hydrogel, which is characterized by comprising the following steps:
(1) Digesting the pig decellularized small intestine submucosa powder with digestive juice, centrifuging, collecting supernatant, and adjusting pH to 7-7.5; adding salt, separating out, centrifuging for several times, retaining precipitate, and freeze drying;
(2) Adding acetic acid with v/v of 0.1-0.2% into dried precipitate powder at a concentration of 50-100mg/ml to dissolve, and regulating osmotic pressure to physiological level with DMEM;
(3) Ultrasound is carried out on the silk fibroin solution with w/v of 3-6%, and the solution obtained in the step (2) is added into the silk fibroin solution after ultrasound according to the volume ratio of 1:1;
(4) Ultrasonic inoculating the mixed solution into a pore plate, and incubating to obtain the compound;
The pig decellularized small intestine submucosa powder is prepared by degreasing, decellularizing, pepsin digestion, repeated salting-out centrifugation, vacuum freeze drying, crushing and grinding the small intestine submucosa;
The degreasing is that the submucosa of the small intestine is placed in a solution with the volume ratio of methanol to chloroform of 1:1-1:1.5 in a dark place;
the decellularization is to soak the defatted pig small intestine submucosa in solution of 0.04-0.06% trypsin/0.04-0.06% disodium ethylenediamine tetraacetate with w/v of 0.04-0.6% and shake in SDS and physiological saline with w/v of 0.4-0.6% for 4-5 hours;
The digestive juice in the step (1) is pepsin digestive juice, the mass ratio of the pig decellularized small intestine submucosa powder to the pepsin digestive juice is 9:1-11:1, acetic acid with v/v of 2.5% -3.5% is added according to 9.5-10.5mg/ml, and after 48-72h, the pig decellularized small intestine submucosa has no floccule, is semitransparent, and is centrifuged.
2. The silk fibroin-binding extracellular matrix hydrogel of pig decellularized intestinal submucosa prepared by the preparation method of claim 1.
3. Use of the silk fibroin-bound porcine decellularized small intestine submucosa extracellular matrix hydrogel of claim 2 in the preparation of a skin injury repair dressing.
4. Use of the silk fibroin-binding porcine decellularized small intestine submucosa extracellular matrix hydrogel of claim 2 in the preparation of biocompatible cell scaffolds.
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