CN109481737A - Bionical double-deck dressing of one kind and preparation method thereof - Google Patents
Bionical double-deck dressing of one kind and preparation method thereof Download PDFInfo
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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Abstract
本发明提供了一种仿生双层敷料，包括脱细胞动物小肠粘膜下层和交联在膜层上的凝胶层，所述膜层与凝胶层共同构成呈双层结构的仿生双层敷料；其制备步骤为包括脱细胞小肠粘膜下层制备、脱细胞小肠粘膜下层粉末制备、脱细胞小肠粘膜下层溶液制备和仿生双层敷料制备步骤，将、脱细胞小肠粘膜下层溶液与交联液混合后加至脱细胞小肠粘膜下层表面，冷冻交联后得到仿生双层敷料。本发明制得的仿生双层敷料不仅具有优异的抗张性能，而且具有半透屏障，能够防止水分蒸发、细菌穿透。同时，本发明制备的仿生双层敷料有利于创面的愈合，能够显著提高创面的愈合速度，且术后第3天和第7天创面愈合率明显加快。
The invention provides a biomimetic double-layer dressing, comprising a decellularized animal small intestine submucosa and a gel layer cross-linked on the membrane layer, the membrane layer and the gel layer together constitute a biomimetic double-layer dressing with a double-layer structure; The preparation steps include the preparation of decellularized small intestine submucosa, the preparation of decellularized small intestine submucosa powder, the preparation of acellular small intestine submucosa solution and the preparation of biomimetic double-layer dressing. The biomimetic double-layer dressing was obtained after freezing and cross-linking to the surface of the submucosa of the decellularized small intestine. The biomimetic double-layer dressing prepared by the invention not only has excellent tensile properties, but also has a semi-permeable barrier, which can prevent water evaporation and bacterial penetration. At the same time, the biomimetic double-layer dressing prepared by the invention is beneficial to the healing of the wound surface, which can significantly improve the healing speed of the wound surface, and the wound healing rate is obviously accelerated on the 3rd and 7th days after the operation.
Description
Technical field
The invention belongs to human skin tissue engineering material field, specifically a kind of bionical double-deck dressing and its preparation side
Method.
Background technique
Human skin is made of epidermis, corium, three layers of subcutaneous tissue.Epidermis is used to prevent moisture, Electrolyte Leakage, pathogeny
It is invaded in microorganism and chemical substance, while being breathed, being perspired by skin accessory organ；Corium is made of fiber, matrix and cell,
Matrix is mainly that cell provides physical support and metabolism place；Subcutaneous tissue is located at corium lower part, by loose connective tissue
It is formed with fat lobule, for preventing heat dissipation, energy reserve and resisting external mechanicalness impact.
Bionical bilayer dressing, can be referred to as artificial skin or bionics skin, mainly using Method of Tissue Engineering to different
Kind tissue carries out de- cell, modified remodeling into the biological support with double-layer structure, then trains to epidermal cell, fibroblast
It supports and expands, and recombinated after so that its cell is reached certain amount.The structure of bionical bilayer dressing is essentially identical to the mankind's
Skin histology, comprising can angling stratified squamous epithelium (surface layer) and (deep layer) containing fibroblastic connective tissue, it has
The partial function of human normal skin, suitable for repairing the skin often resulted in by situations such as burn, wound, diabetes, chronic ulcer
Skin wound.
Currently, it is mostly that spongy dermis scaffold or de- cell are raw that organizational project, which prepares the material that the bionical double-deck dressing uses,
Object film, because it lacks the mechanical performance of certain resistance to compression and anti-drawing, using limited.The ideal bionical double-deck dressing should have table
Skin, the biomimetic features of corium and certain mechanical performance, and should have the characteristics that it is not easily broken, can suture, facilitate operation.
Studies have shown that animal sources extracellular matrix plays an important role in organizational project Regeneration and Repair, at grouping
It is close at, institutional framework and human skin tissue, and animal sources cell origin is abundant, securely and reliably, extracts convenient for industrialization, is
A kind of good regenerating tissues stock support.Research is it is also shown that submucous layer of small intestine (small intestinal
Submucosa, abbreviation SIS) as a kind of selective semi-permeable membrane, have good mechanical performance, histocompatbility and lower
Immunogenicity, it is similar with epidermis function, it can be used as a kind of natural extracellular matrix material.But natural SIS is film-form
Material, the reparation for tissues such as skins lack enough thickness, hole and water absorption rate.Display is had been reported that, using the master of SIS
Component collagen albumen is wanted to prepare hydrogel, slow-released carrier and 3D cell culturing bracket as cell and drug, but hydrogel object
Rationality can be poor, and matter is crisp frangible, inconvenient, therefore, limits hydrogel in the clinical application of skin wound reparation.
In addition to this, CN101361990B discloses a kind of double-layered artificial skin, includes fibroblast, it is by taking off
The membranaceous bio-derived material of cell by fibroblast and its synthesizes the extracellular matrix and cell growth factor secreted as surface layer
Son, which is compound in inside biologic bracket material, forms skin corium, and the two is chimeric to be constituted；The membranaceous bio-derived material of de- cell
Membranaceous bio-medical material, including de- cell submucous layer of small intestine, de- cell are formed by through de- cell for natural biological tissue
Dermal matrix, de- cell fascia, de- cell submucous layer of bladder, de- cell amnion, de- cell are dural any；Described
Fibroblast includes the stem cell that can break up to fibroblast；The biologic bracket material is to grow to provide for cell
The backing material of three-dimensional space, including fibrinogen, fibrin, alginate, collagen, hyaluronic acid, sulfuric acid are soft
Any or several mixing of ossein, chitosan, hydrogel, gelatin, polylactic acid, polyglycolic acid.
Double-layered artificial skin disclosed in CN101361990B uses animal source cell for raw material, although to a certain degree
On can promote the regeneration of surface of a wound skin, skin elasticity, flexibility and the mechanical endurance after enhancing wound healing reduce scar and increase
It is raw, control contracture.But its preparation process is complicated, and incubation time is long, at least needs for artificial skin to be placed in culture solution and cultivates 9
It.
Summary of the invention
It is of the invention the object of the present invention is to provide a kind of bionical double-deck dressing based on problems of the prior art
Another object is to provide a kind of preparation method of bionical double-deck dressing.
As known to those skilled in the art, for skin tissue engineering, skin histology preparation usually requires prolonged external
Culture, and in vitro culture is higher to condition of culture and environmental requirement, belongs to the technical problem of skin tissue engineering.The present invention is with dynamic
Object small intestine is raw material, and the bionical double-deck dressing prepared by specific technique, distinctive ingredient and structure are directly nutrients
The exchange of matter and the migration of vascular endothelial cell provide good condition, without just can be used for carefully after carrying out cultured ex vivo for several days
Born of the same parents' transplanting, solves the technical problem of prolonged in vitro culture.
Unless otherwise specified, raw material of the present invention is commercial product as known to those skilled in the art, the percentage
Than being mass percent.
The object of the present invention is achieved like this:
A kind of bionical double-deck dressing, the gel layer including film layer and crosslinking in film layer, the film layer are collectively formed with gel layer
Bionical bilayer dressing, the film layer are de- multicellular animal submucous layer of small intestine, and the gel layer is with animal submucous layer of small intestine
For freezing gel made from raw material；
The freezing gel can be made as follows:
Step 1: first removing the placenta percreta and muscle layer of animal mucous membrane of small intestine, degreasing, de- cell processing are then carried out to it, is taken off
Multicellular animal submucous layer of small intestine；
Step 2: will first state gained animal submucous layer of small intestine and crush, then by smashed de- multicellular animal submucous layer of small intestine
It is filtered after enzymatic hydrolysis, gained filtrate, which is saltoutd, takes its precipitating after centrifugal treating, then will dialyse after gained precipitating acetate dissolution
Processing obtains animal submucous layer of small intestine sponge after the freeze-drying of gained dialyzate, then gained animal submucous layer of small intestine sponge is ground
Animal submucous layer of small intestine powder (SIS powder) is obtained after mill；
Step 3: first aforementioned SIS powder being dissolved in ethanesulfonic acid or phosphate, obtains SIS solution, then by SIS solution and crosslinking
Liquid mixing obtains mixed system, then aforementioned mixed system is added under the de- multicellular animal mucous membrane of small intestine after buffer impregnates
Layer surface, and carry out freezing processing to it is cross-linked to form after freezing de- multicellular animal submucous layer of small intestine surface is chilled
A kind of colloid substance, i.e. freezing gel；
According to an embodiment of the invention, it is pig, ox, sheep, horse or monkey that above-mentioned animal mucous membrane of small intestine, which is animal mucous membrane of small intestine,
One of mucous membrane of small intestine；Wherein, pig intestinal mucosa is preferred.
It is found in research, gel layer has a major impact cellular material metabolism, merely uses fibrinogen, fiber egg
As gel layer, permeability and tensile property are poor for white, alginate, collagen, hyaluronic acid or hydrogel.The present invention
The micropore being mutually communicated is distributed on the bionical double-deck dressing gel layer of preparation, and micropore average pore size is 50-400 μm, can be promoted
Into cytotrophy mass exchange and migration of vascular endothelial cells, and it contains the raised growth factor, can promote local cellular proliferation.
It has also been found that, merely using the preferable freezing gel of compressive property in the present invention, tensile strength, especially covering are big in research
The tensile strength of area surface of a wound material requested is not good enough, and bacterial barriers performance is also undesirable, cannot be used directly for skin wound reparation,
Freezing gel and de- multicellular animal submucous layer of small intestine are combined together by the present invention by specifically method, are solved existing solidifying
The technical problem of glue-line, bionics skin tensile property and bacterial barriers performance difference.
For the structure for advanced optimizing the bionical double-deck dressing, its comprehensive performance, the film layer of the above-mentioned bionical double-deck dressing are promoted
With a thickness of 80-100 μm, gel layer thicknesses are 180-220 μm.
De- multicellular animal submucous layer of small intestine of the present invention is referred to as SIS film, the de- cell submucous layer of small intestine powder
End is referred to as SIS powder, and the de- cell submucous layer of small intestine solution is referred to as SIS solution.
The preparation method of the bionical double-deck dressing as described above, including SIS film preparation, the preparation of SIS powder, the preparation of SIS solution
With the bionical double-deck dressing preparation step, specifically:
Step 1, SIS film preparation: taking fresh chitterlings, first removes placenta percreta and muscle layer；Then volume ratio=1.2- is placed it in
It impregnates 6-24 hours in the methanol and chloroform mixed liquor of 2:1, and is rinsed with deionized water；Then by the chitterlings after aforementioned rinsing
It is placed in the mixed system of 0.025-0.1% pancreatin and 0.05-0.1% ethylenediamine tetra-acetic acid and digests at room temperature 8-15 hours；
And it washes with water；The chitterlings after the cleaning of aforementioned water are placed in the physiological saline containing 0.5% lauryl sodium sulfate again and are vibrated
6-24 hours, obtain SIS film；It is saved backup after gained SIS film normal saline flushing；
Step 2, prepared by SIS powder: will crush after aforementioned gained SIS film acetic acid soaked overnight, is placed in enzyme in pepsin
It is filtered after solution, gained filtrate takes its precipitating after 1-4 times is saltoutd centrifugation；Then by gained precipitating be dissolved in acetic acid, and with dialyse
Bag dialysis 15-72 hours, obtains dialyzate；Gained dialyzate is lyophilized again, obtains spongy SIS, it is finally that gained is spongy
SIS grind into powder obtains SIS powder；Gained SIS powder, which is placed in centrifuge tube, to be saved backup；
Step 3, prepared by SIS solution: gained SIS powder is dissolved in the 2- (N- morpholine) of pH=3-7 by final concentration 8-20mg/ml
In ethanesulfonic acid or phosphate, SIS solution is obtained after mixing evenly under the conditions of 3-8 DEG C；
Step 4, the bionical double-deck dressing preparation: take aforementioned gained SIS film be placed in buffer impregnate 10-24 hours after be laid in training
It supports in ware, suck dry moisture；It takes aforementioned gained SIS solution to mix with crosslinked fluid, mixed system is obtained after stirring；By gained mixture
The SIS film surface in culture dish is added in system, is placed in subzero 20 DEG C and freezes 15-30 hours into subzero 180 DEG C of environment, room temperature
The bionical double-deck dressing is obtained after dissolving in environment.
Preferably, in the preparation method of the above-mentioned bionical double-deck dressing, the buffer is hydrochloric acid solution or 2- (N- morphine
Quinoline) ethanesulfonic acid, the crosslinked fluid is glutaraldehyde or tea polyphenols and phosphatic mixed solution or 1- ethyl -3- (3- diformazan ammonia third
Base) carbodiimide hydrochloride solution and HOSu NHS mixed solution；
It is highly preferred that the buffer is hydrochloric acid solution, the crosslinked fluid is that glutaraldehyde or tea polyphenols are mixed with phosphatic
Close solution.
For the structure for further optimizing the bionical double-deck dressing, its comprehensive performance is promoted, according to an embodiment of the invention,
The final concentration of 0.075-1% of the glutaraldehyde, the final concentration of 0.5-4g/L of the tea polyphenols.
The present invention has following beneficial effect
The present invention uses animal mucous membrane of small intestine for raw material, by specific technique, is carbonized using water-soluble zero-length crosslinking reagents
Diimine forms amido bond by the aminoterminal and c-terminus of collagen in the case where not being grafted any chemical group, chilled
One layer of freezing gel is formed on animal submucous layer of small intestine surface afterwards, realizes the preparation of the bionical double-deck dressing.
The bionical double-deck dressing produced by the present invention not only has excellent tensile property, tensile strength up to 1.26MPa, and
And there are semi-permeable barriers, moisture evaporation, bacterial penetration can be prevented.Meanwhile the bionical double-deck dressing prepared by the present invention is conducive to
The healing of the surface of a wound, can significantly improve the healing rate of the surface of a wound, and postoperative 3rd day and the 7th day Wound healing rate are obviously accelerated.
Using the bionical double-deck dressing of present invention process preparation, cell can be made sufficiently to adhere to after incubated in vitro 2h, and
Cell slow-released carrier is constructed, can be directly used for dermatoplasty, and its distinctive ingredient and structure are directly intracellular nutritional substance
Exchange and the migration of vascular endothelial cell provide good condition.In vitro culture number is needed compared to existing common bionics skin
It just can be used for cell transplantation after it, the present invention needs the technology of long-time in vitro culture difficult before solving existing bionics skin transplanting
Topic.In addition, compared to the preparation process of double-layered artificial skin in CN101361990B, the bionical double-deck dressing preparation process of the present invention
Only need can be completed within 5 days, it is time-consuming shorter.
The upper layer of the bionical double-deck dressing of the present invention is film layer, and lower layer is freezing gel, outside main component and n cell
Matrix components are close, have many advantages, such as anti-drawing, anti-shrink, anti-clamp, anti-extrusion.Compared to existing bionics skin, the present invention
Bionical bilayer dressing elastoresistance deformability is quite excellent, even if cell is added dropwise after by squeezing or absorbing the moisture in skin
Suspension can be such that it restores rapidly, while being capable of quick adsorption cell.
The bionical double-deck dressing of the present invention realize in freezing gel layer it is porous be mutually communicated, there is excellent formative memory
Function, and being capable of active resilient adherent cell suspension when cell inoculation.
The bionical double-deck dressing of the present invention is degradable biomaterial, safe and non-toxic, has good biological property, especially
Water absorption rate is strong, good biocompatibility, and degradation in vitro is high, and dermatoplasty success rate is high, and it contains the raised growth factor, can promote
It is grown into local cells, hyperblastosis.
Detailed description of the invention
Fig. 1: the SIS film prepared in the embodiment of the present invention 1；
Fig. 2: SIS film is laid in culture dish bottom in the embodiment of the present invention 1；
Fig. 3: in the embodiment of the present invention 1 when the mixed system of SIS film surface addition SIS solution and crosslinked fluid；
Fig. 4: in the embodiment of the present invention 1 after the mixed system of SIS film surface addition SIS solution and crosslinked fluid；
Fig. 5: bionical bilayer dressing in the embodiment of the present invention 1 (before cleaning)；
Fig. 6: bionical bilayer dressing gel layer in the embodiment of the present invention 1；
Fig. 7: bionical bilayer dressing in the embodiment of the present invention 1 (after cleaning)；
Fig. 8: the film layer structure of bionical bilayer dressing in the embodiment of the present invention 1；
Fig. 9: the gel layer structure of bionical bilayer dressing in the embodiment of the present invention 1；
Figure 10: bionical bilayer dressing cross section structure in the embodiment of the present invention 1；
Figure 11: in the embodiment of the present invention 1 before bionical bilayer dressing inoculating cell；
Figure 12: bionical bilayer dressing inoculating cell prepares in the embodiment of the present invention 1；
Figure 13: it is added dropwise after cell suspending liquid at once at once after the bionical double-deck dressing inoculating cell of the embodiment of the present invention 1；
Figure 14: bionical double-deck dressing bacterium (Escherichia coli, staphylococcus aureus) the penetration test result of the embodiment of the present invention 1
(means standard deviation), A Escherichia coli, B staphylococcus aureus (* P < 0.05, n=4)；Figure 15: the embodiment of the present invention 1 is imitative
The mechanical property of raw bilayer dressing, A tensile strength, B elongation at break, C Young's modulus, (* p < 0.05, n=3)；
Figure 16: the moisture-vapor transmission WVTR (* * p < 0.01, n=3) of the bionical double-deck dressing of the embodiment of the present invention 1；
Figure 17: mouse healing postoperative wound surface situation of the present invention；
Figure 18: mouse healing postoperative wound surface rate (* P < 0.01, n=3) of the present invention.
Specific embodiment
The present invention is specifically described below by specific embodiment, it is pointed out here that following embodiment is served only for this hair
It is bright to be further described, it should not be understood as limiting the scope of the invention, the person skilled in the art of this field can root
Some nonessential modifications and adaptations are made to the present invention according to foregoing invention content.
Embodiment 1
A kind of preparation method of the bionical double-deck dressing, is made according to following steps:
Step 1, SIS film preparation: taking fresh chitterlings, first passes through mechanical means removal placenta percreta and muscle layer；Have in the present embodiment
Body method are as follows: first fresh pig small intestine contents are squeezed out, cut into about 15cm long segment, remove the mesenterium for being attached to surface,
It is placed in rustless steel container, tap water rinses for several times；Chitterlings after aforementioned cleaning are laid on plate, to cause the suitable of the right side from left
Sequence strikes off mucous membrane of small intestine layer, placenta percreta and muscle layer repeatedly.Until translucent submucous layer of small intestine appears；Again repeatedly through tap water
It rinses and small intestine is run through into overturning, further scraping off residual tissue with chopsticks afterwards for several times；Finally splitted into scissors along the small intestine longitudinal axis
The fritter of about 10cm × 15cm, after tap water repeated flushing, distilled water rinses 3 times and obtains trees-Osima jacoti, Osima excavata.Then will
It is placed in volume ratio=1.2:1 methanol and chloroform mixed liquor soak degreasing 12 hours, and is rinsed 3 times with deionized water；So
The chitterlings after aforementioned rinsing are placed in afterwards in the mixed system of 0.05% pancreatin and 0.07% ethylenediamine tetra-acetic acid at room temperature
Digestion 12 hours；And it is cleaned 3 times with pure water；The chitterlings after the cleaning of aforementioned water are placed in containing 0.5% dodecyl sulphate again
It is vibrated 6 hours in the physiological saline of sodium, obtains SIS film, as shown in Figure 1；Gained SIS film is placed in 4 with normal saline flushing 3 times
DEG C environment in save backup；
Step 2, prepared by SIS powder: beater (model: AUX will be used after 0.5% acetic acid soaked overnight of aforementioned gained SIS film
HX-PB1018, Chinese Guangdong) it is crushed, gained powder and pepsin (it is public to be originated from the extensive and profound in meaning star biotechnology responsibility in Beijing
Department) in mass ratio=100:1 mixing, at room temperature digest 2 days after filter, gained filtrate saltoutd twice centrifugation after take it
Precipitating；Then gained precipitating is dissolved in 0.1% acetic acid, and is dialysed 24 hours with 8KD (8000 dalton) bag filter, obtained
Analyse liquid；Gained dialyzate is lyophilized with freeze drier (model: Coolsafe TM 55&110-4L/55-9L, Denmark) again, is obtained
To spongy SIS, finally gained spongy SIS analysis grinder (model: IKA A11basic, Germany) is pulverized
End obtains SIS powder；Gained SIS powder is placed in 50ml (milliliter) centrifuge tube, is saved backup in 4 DEG C of environment；
Step 3, prepared by SIS solution: aforementioned gained SIS powder is dissolved in pH=5's by final concentration 10mg/ml (mg/ml)
In 2- (N- morpholine) ethanesulfonic acid, it is evenly stirred until under the conditions of 4 DEG C after being completely dissolved and obtains SIS solution；
Step 4, it the bionical double-deck dressing preparation: takes aforementioned gained SIS film to be placed in the hydrochloric acid solution of PH=3 overnight, is put down after taking-up
(as shown in Figure 2), suck dry moisture are laid in culture dish；Taking aforementioned gained SIS solution with crosslinked fluid, (crosslinked fluid is matched in the present embodiment
Method processed: the phosphoric acid solution of pH=7 is mixed with glutaraldehyde (glutaraldehyde volume fraction is 2.5-5%) according to volume ratio=4:1,
That is the final concentration of 0.5-1% of glutaraldehyde) it is mixed according to molar ratio=2:1, mixed system is obtained after stirring；By gained mixed system
The SIS film surface (as shown in Figure 3, Figure 4) being uniformly added into culture dish is placed in -100 DEG C of environment and freezes 24 hours, room
Obtain the bionical double-deck dressing (as shown in Figure 5) after dissolving in warm environment, in this step, the glue of trees-Osima jacoti, Osima excavata surface formation
Shape substance is freezing gel, and freezing gel and de- cell trees-Osima jacoti, Osima excavata form the bionical double-deck dressing after being cross-linked with each other,
As shown in Figure 6.The thicknesses of layers of the bionical double-deck dressing of gained is about 100 μm, and gel layer thicknesses are 200 μm, average pore size 120
μm。
The bionical double-deck dressing of gained first uses 0.1mol (mole) disodium phosphate soln to rinse 2 hours, changes liquid within every 30 minutes
Once；2h wash with distilled water again, changes the liquid once for every 30 minutes, and the bionical double-deck dressing after cleaning is as shown in Figure 7；Then by it
It is dipped in PBS (phosphate buffered saline solution (phosphate buffer saline)), is saved backup in 4 DEG C of environment.
Embodiment 2
A kind of preparation method of the bionical double-deck dressing, is made according to following steps:
Step 1, SIS film preparation: taking fresh small sheep intestines, first passes through mechanical means removal placenta percreta and muscle layer；Then it places it in
Soak degreasing 15 hours in volume ratio=1.5:1 methanol and chloroform mixed liquor, and rinsed 3 times with deionized water；Then will before
Small sheep intestines after stating rinsing are placed in 0.08% pancreatin and the mixed system of 0.1% ethylenediamine tetra-acetic acid that digest 10 at room temperature small
When；And it is cleaned 3 times with pure water；The small sheep intestines after the cleaning of aforementioned water are placed in the physiology containing 0.5% lauryl sodium sulfate again
It is vibrated 8 hours in salt water, obtains SIS film；Gained SIS film is placed in 4 DEG C of environment and is saved backup with normal saline flushing 3 times；
Step 2, prepared by SIS powder: will be crushed after 0.5% acetic acid soaked overnight of aforementioned gained SIS film with beater, institute
Powder and pepsin in mass ratio=100:1 mixes, enzymatic hydrolysis filters after 2 days at room temperature, and gained filtrate is through twice
It saltouts and takes its precipitating after being centrifuged；Then gained precipitating is dissolved in 0.1% acetic acid, and saturating with 8KD (8000 dalton) bag filter
Analysis 30 hours, obtains dialyzate；Again by gained dialyzate freeze drier (Coolsafe TM 55&110-4L/55-9L, pellet
Wheat) freeze-drying, spongy SIS is obtained, finally by gained spongy SIS analysis grinder (model: IKA A11basic, Germany)
Grind into powder obtains SIS powder；Gained SIS powder is placed in 50ml centrifuge tube, is saved backup in 4 DEG C of environment；
Step 3, prepared by SIS solution: aforementioned gained SIS powder is dissolved in the phosphate solution of PH=7 by final concentration 15mg/ml
In, it is evenly stirred until under the conditions of 6 DEG C after being completely dissolved and obtains SIS solution；
Step 4, it the bionical double-deck dressing preparation: takes aforementioned gained SIS film to be placed in the hydrochloric acid solution of PH=3 overnight, is put down after taking-up
It is laid in culture dish, suck dry moisture；Aforementioned gained SIS solution and crosslinked fluid is taken (to be crosslinked liquid making method in the present embodiment: by pH
=7 phosphoric acid solution is mixed with tea polyphenols (concentration 10-20g/L) according to volume ratio=4:1, i.e. the final concentration of 2- of tea polyphenols
It 4g/L) is mixed according to molar ratio=1.5:1, obtains mixed system after stirring；Gained mixed system is uniformly added into culture dish
SIS film surface, be placed in -105 DEG C of environment and freeze 18 hours, the bionical double-deck dressing is obtained after dissolving in room temperature environment.
The thicknesses of layers of the bionical double-deck dressing of gained is about 95 μm, and gel layer thicknesses are 210 μm, and average pore size is 105 μm.This step
In, the colloid substance that small sheep intestines submucosa surface is formed is freezing gel.
The bionical double-deck dressing of gained first uses 0.1mol disodium phosphate soln to rinse 2 hours, changes the liquid once within every 30 minutes；Again
2h wash with distilled water is changed the liquid once for every 30 minutes；Then it is dipped in PBS (phosphate buffered saline solution), in 4 DEG C of environment
In save backup.
Embodiment 3-8
The bionical double-deck dressing is prepared according to technological parameter in table 1 referring to embodiment 1 or embodiment 2；
Technological parameter in 1 embodiment 3-8 of table
Electron-microscope scanning test
Experimental design: the bionical double-deck dressing prepared in the sampling embodiment of the present invention 1 carries out electron-microscope scanning examination as sample
Test, specifically: sample is cut to 1cm × 1cm, brittle fracture is carried out to its sample using liquid nitrogen quenching method, first 50%,
75%, be dehydrated 5 minutes in 85%, 95%, 100%, 100% concentration gradient alcohol respectively, then 50%, 75%, 85%,
95%, it is dehydrated respectively 5 minutes in 100%, the 100% concentration gradient tert-butyl alcohol, then cross-section face upward of sample is sticked to metal tray
On；And the metal spraying on sample carrier, by Electronic Speculum observe sample section, electron-microscope scanning film layer structure (epidermis), as shown in Figure 8；
Electron-microscope scanning gel layer structure (skin corium), as shown in Figure 9；Its cross section structure of electron-microscope scanning, as shown in Figure 10.
Interpretation of result: as shown in Figure 8, the bionical double-deck dressing film layer of the present invention is in threadiness；As shown in Figure 9, the present invention is imitative
Irregular micropore is distributed on raw bilayer dressing gel layer, studies have shown that the micropore may advantageously facilitate nutriment in cell
Exchange and vascular endothelial cell migration；As shown in Figure 10, the bionical double-deck dressing film layer of the present invention is cross-linked with each other with gel layer,
Film layer has combined together with gel layer, shows that the stability of the bionical double-deck dressing of the present invention is preferable.
The test of cell adsorption capacity
Experimental design: taking the bionical double-deck dressing prepared in the embodiment of the present invention 1 is sample, and PBS is added in sample to be made
It is totally submerged, and soaked overnight makes it sufficiently absorb water to be placed on plate, as shown in figure 11, gel layer upward, film layer (SIS
Layer) downward；It squeezes the bionical double-deck dressing and removes its moisture with filter paper, make it in flat-shaped, as shown in figure 12；By cell suspending liquid
The case where being added dropwise in the bionical double-deck dressing gel layer surface, observing the bionical double-deck dressing adherent cell suspension, it is outstanding to be added dropwise cell
After supernatant liquid at once as shown in figure 13.
Interpretation of result: as seen from Figure 11, before inoculating cell, being full of liquid in bionical bilayer dressing, sparkling and crystal-clear full
It is full；As seen from Figure 12, after applying pressure to it, moisture is excluded, and bionical bilayer dressing is shunk in flat；By Figure 13
As can be seen that after gel layer surface drop cell suspending liquid, the bionical double-deck dressing can rapid adherent cell suspension, appearance
It is sparkling and crystal-clear full.It can be seen that the bionical double-deck dressing cell adsorption capacity of the present invention is strong.
Bacterial penetration test
Experimental design:
(1) respectively by freezing gel (SIS gel) in the present embodiment 1, embodiment 2, the bionical double-deck dressing (SIS bilayer),
SIS film (SIS membrance) and gelfoam (Gelatin aponge, thick 1mm, Nanjing Jinling Pharmaceutical Factory), single layer is sterile
Petrolatum gauze (Vaseline gauze), is cut to 10mm diameter circular, material is given Co60 radiation sterilization, 3kGy is total to three times；
(2) 1 × 10 is configured8Colony Forming Unit (colonyforming units, CFU)/ml staphylococcus aureus, large intestine
Bacillus bacterium solution.The four groups of materials and sterile vaseline gauze of same size will be cut into, totally five groups, is put in high concentration agar culture
20 μ l bacterium solutions are added dropwise in material center in ware, notice that, not beyond material ranges, 37 DEG C of constant temperature incubators are cultivated 24 hours；
(3) agar below material is dug out with sterile razor blade, is put in the sterile centrifugation tube for containing 10ml pbs, PBS shakes repeatedly
Swing cleaning agar block surface, separation of bacterial；
(4) after the PBS containing bacterium in test tube being carried out doubling dilution, it is routinely applied to M-H drug sensitive test agar plate, is put into incubator 37
DEG C, constant temperature incubation 12 hours；
(5) according to bacterial colony count principle, bacterial number in PBS solution is calculated.
Interpretation of result:
Bacterium is colonized the quantity on agar plate by SIS material, gelfoam or petrolatum gauze, and it is real to carry out bacteriology
It tests, discovery penetrates S. aureus L-forms bacterial number and Escherichia coli in embodiment 1 by the bionical double-deck dressing of pig intestinal mucosa preparation
Quantity is minimum (as shown in figure 14), and average value is 4.63 × 104CFU and 49.50 × 104CFU (Colony Forming Unit, colony
Forming unit), substantially less than other groups (P < 0.05, n=4)；The bacterium amount that Escherichia coli penetrate freezing gel is maximum, puts down
Mean value is 480.75 × 104CFU, except each group outer compared with petrolatum gauze has statistical difference (P < 0.05, n=4).Gold
The bacterium amount of Portugal's bacterium bacterial penetration petrolatum gauze is maximum, and average value is 68.13 × 104CFU, bionical bilayer dressing group and SIS film
Group more has statistical difference (P < 0.05, n=4).
Mechanical property test
Experimental design: preparation a variety of materials are cut into dumbbell shaped diaphragm, and use 5567 testing of materials system of Instron
Unified test tries its mechanical performance, is divided into SIS bilayer (MS), SIS film (M), and commercialization 1mm thickness gelatin (G).Sample is fixed
On specimen holder, sample is stretched according to the rate of extension of 50mm/min, until it is pulled off completely, machine is automatically recorded and analyzed
As a result.Duplicate measurements 3 times.
In experimentation SIS freezing gel can not clamp, after clamp can fragmentation, be unable to test this performance.
Interpretation of result: as shown in Figure 15, the Young's modulus of bionical bilayer dressing (MS) is in the embodiment of the present invention 1
10.95MPa, hence it is evident that it is higher than commercialization gelfoam 0.93MPa, meanwhile, tensile strength 1.26MPa, elongation at break is
35.41%, it is significantly higher than commercialization gelfoam 0.09MPa and 16.06%.(* P < 0.05, n=3), sufficiently shows its anti-tensile
It has excellent performance.
Water vapour permeability test
Experimental design: bionical bilayer dressing (being denoted as MS), freezing gel in the embodiment of the present invention 1 is taken (to be denoted as S), SIS film
(being denoted as M) and commercialization gelfoam (being denoted as G), carries out the test of moisture-vapor transmission (WVTR), referring to ASTM standard, i.e.,
ASTM American society for testing and materials (American Society of Testing Materials) standard.First by each group material
Material sample is cut into diameter and is the circular film of 35mm, and covers diameter 34mm with it, fills the measuring cup of 10ml water.Rim of a cup is close
Whole device is put into 50% relative humidity, in 37 DEG C of incubator, is once claimed to device within test macro every 2 hours by envelope
Weight, continuous weighting are tested 24 hours.Period related data is automatically recorded and is analyzed by tester, duplicate measurements 3 times, obtains its mechanics
Performance and moisture-vapor transmission the result is shown in Figure 16.
Interpretation of result: as shown in Figure 16, the moisture-vapor transmission of bionical bilayer dressing (MS) in the embodiment of the present invention 1
It (WVTR) is respectively 978g/m2/ day, 1358g/m2/ day (p < 0.01, n=3), shows the performance of its water vapor transmitance
It is good.
The test of cell in vitro compound criteria
Experimental design
The originally culture of GFP mouse BMSCS, the specific method is as follows:
(1) GFP young rat is taken out from mouse cage, is placed it in beaker and is impregnated 5 minutes with 75% alcohol, then in superclean bench
It is interior to be clamped with aseptic nipper to the culture dish containing sterile PBS；Sterile PBS cleaning removes alcohol, puts again after PBS solution rinsing twice
Enter another sterile petri dish；
(2) antiseptic hand washing wears sterile gloves, and sterile scissors and tweezers are taken out from instrument container, with scissors and tweezers, by mouse four
Limb cutting, is placed in sterile petri dish containing a small amount of PBS sterile solution, skin is torn off completely, removes claw.The muscle of surrounding
Soft tissue removal only retains exposure long bone of limbs, the sterile culture of another new drying is placed on after long bone is washed with PBS
Ware replaces sterile scissors, shreds bone tissue as far as possible with scissors；
(3) after the configured good dedicated complete medium of mesenchymal stem cell of 5ml is added after bone tissue shreds, sterile glass is used
Suction pipe piping and druming mixing, is transferred into 25cm after mixing2Aseptic plastic culture bottle, bottle cap are tightened, and are sterilized and are cultivated with alcohol wipe
Bottle simultaneously places it in 37 DEG C, the CO of gas fraction 5%2Incubator changes liquid afterwards for 24 hours and removes suspension cell and tissue block, later
Change liquid within every 2-3 days；
(4) under the microscope, cell fusion degree is expanded up to being passed on after 80% by trypsin digestion, can be passed within each 3-4 days
Once.
Cell passage
(1) culture solution before in the culture bottle for having covered with cell is outwelled；
(2) 2mlPBS is added to clean 2 times, the digestion of 0.25% trypsin solution of 1ml is added；
(3) bottle cap is covered, is placed under inverted microscope and observes.Over time, before adherent cell is gradually rounded shape,
Pancreatin is outwelled in cell also non-levitating, 10ml culture solution is added, digestion is allowed to terminate；
(4) it being blown and beaten repeatedly with suction pipe for several times, adherent cell is blown and beaten as suspension, is sucked in centrifuge tube, 500rpm is centrifuged 5min,
It abandons after supernatant plus 3ml complete medium is resuspended cell and carries out cell count, then cell is diluted to 3 × 105A/ml's is close
Degree takes 1ml that 75cm is added2In culture bottle, then 3ml complete medium is added, gently shakes culture bottle, be evenly distributed on cell
In culture bottle, 4 culture bottles are divided into, put 37 DEG C, the CO of gas fraction 5%2Cell culture incubator is incubated overnight.
Cell in vitro plantation
(1) by freezing gel, the bionical double-deck dressing, SIS film and gelfoam (thick 1mm, Nanjing Nanjing in the embodiment of the present invention 1
Pharmaceutical factory) using punch cut into diameter be the discoid of 6mm be placed in culture dish, with 75% ethyl alcohol impregnate 1h, sterile PBS
It rinses 5 times；
(2) another culture dish is taken, plate-like material is put into.The DMEM culture solution that appropriate 10% fetal calf serum is added floods it completely
Not yet, 37 DEG C, the interior hatching of constant temperature incubator is for 24 hours；
(3) third generation GFP Marrow Mesenchymal Stem Cells, cell count plate count after 0.5% trypsin digestion.Cell
500rpm is centrifuged 5min after digestion, abandons after supernatant plus is resuspended after the DMEM medium centrifugal of 5ml10% fetal calf serum, and cell is dilute
It releases, adjustment concentration to 8 × 104/ml；
(4) material prepared before is taken out from incubator, is placed in super-clean bench, aseptic nipper clamping is transferred to 96 orifice plates
In, it is put in bottom；
(5) 100 μ l cell suspensions (i.e. every hole cell number is 8000) is added in every hole, is grouped every group 5 again according to experimental material
Hole is inoculated with 96 orifice plates, without material is control wells only plus containing cell culture medium, and it is cell-free for blank well to contain only culture medium, most
PBS is all added in the hole of outer ring, puts cell incubator and is incubated overnight；
(6) the 5%CO2 constant temperature incubator at 37 DEG C is incubated for, and changes liquid within every 2 days.
CCK-8 absorbance detection
(1) there are culture medium and cell and be control wells without material, only culture medium is blank well, 37 DEG C after inoculation, 5%
CO2Incubator is incubated for；
(2) it transfers the sample on a 96 new orifice plates, to eliminate the influence for the cell being not adhere on bracket；
(3) 15ml centrifuge tube, DMEM culture solution 2.7ml, CCK-8 solution 300ul, stirs and evenly mixs；Mixed solution 100ul, wherein
90ul DMEM culture medium, 10ulcck-8 solution are added to each hole, mix；5%CO2, 37 DEG C of incubation 2h；
(4) culture medium is sucked into 96 new orifice plates, absorbance detection at every hole 100ul, microplate reader 450nm.
Confocal laser scanning microscope (laser scanning confocal microscope, LSCM), it is specific to walk
It is rapid as follows:
Sample is removed to after material culture 1d according to the method described above, excludes the interference of non-adherent cell, PBS is washed 3 times；More than 4%
Polyformaldehyde is fixed for 24 hours；30% sucrose solution dehydrated overnight；Machine upper after material routine film-making is waited for that laser confocal microscope is seen
It examines；The channel green fluorescence (GFP) is selected from outside to inside, to scan 20-30 level along Z axis；Total 50-60um thickness；Scanning is
Cell monolayer, then carry out the synthesis of multi-layer image three-dimensional with laser confocal microscope and rebuild, it is grown in the material with understanding cell
When form and cell space distribution situation in the material.
Interpretation of result
Microscopically observation primary cultured cell
After culture for 24 hours, culture bottle inner wall observes a large amount of tissue blocks, the thin out yellow of culture medium color.Microscopically observation
To a small amount of adherent mesenchymal stem cell, form is irregular, and cell volume is small, is unevenly distributed, will be not adherent when changing liquid
Cell and tissue block removal.After 2-3 days, the growth of colony sample is presented in mesenchymal stem cell, according to cell growth status, often
Change within 2-3 days a not good liquor.Adherent cell proliferation ability gradually becomes by force, and cell increases, is elongated, is in shuttle shape mechanocyte.With
Change liquid, other not adherent cells are removed in culture bottle, and cell can mostly cover with bottom of bottle at 8-9 days, after trypsin digestion, are pressed
It is passed on 2-3 times according to 1:2, it is seen that attached cell form is consistent, is passed within every 3-4 days later.Subsequent experimental is thin using the third generation
Born of the same parents.
The double-deck dressing CCK-8 kit test result
CCK-8 absorbance is measured, the 5th day OD value is greater than SIS the result shows that bionical bilayer dressing group (0.841 ± 0.109)
Film group, gelatin foam group (0.512 ± 0.108,0.443 ± 0.109), compared with freezing gel (0.654 ± 0.147), difference
Not statistically significant, freezing gel group is better than gelatin foam group, compared with SIS film group, no significant difference.At the 7th day
SIS double layer material group (1.011 ± 0.125), be still greater than SIS film and gelatin foam group (0.578 ± 0.104,0.558 ±
0.127), there was no significant difference compared with freezing gel group (0.919 ± 0.130).Freezing gel group OD value be higher than SIS film with it is bright
Gelatin sponge group has significant difference (P < 0.05, n=5).
Confocal laser scanning microscope result: the visible MSCS cell with green fluorescence is in material internal and table under mirror
Widely distributed in face, cell appearance is in shuttle-type, and stretching, extension is good.
Wound healing test
Experimental design
Material prepares: by freezing gel in the embodiment of the present invention 1, bionical bilayer dressing, and SIS film, gelfoam (thick 1mm,
Nanjing Jinling Pharmaceutical Factory) using punch cut into diameter be 10mm square be placed in culture dish, 75% ethyl alcohol impregnate 1h,
Sterile PBS is rinsed 5 times.
The experiment of mouse full thickness dermal
Experiment sets empty five groups altogether, respectively blank control group (control group), freezing gel group (SIS gel),
Bionical bilayer dressing group (SIS bilayer), SIS film group (SIS membrance), gelatin foam group (Gelatin
aponge)。
Experiment specific step is as follows:
(1) preserved skin, by back of mice skin depilatory on the day before formal experiment.1% Nembutal sodium solution is injected intraperitoneally
(0.01ml/g) anesthesia, electric hairdressing device wipe out back of mice hair.Depilatory cream is uniformly applied to back, and 3 to using cotton after five minutes
Ball gently wipes the depilatory cream and hair of back of mice, wipes remaining depilatory cream again to cleaning with warm and humid cotton balls.To mouse
After revival, rearging cage, the raising of row single cage are put back to；
(2) second days, after mouse peritoneal injects 1% Nembutal sodium solution (0.01ml/g) anesthesia, operation was fixed in prone position
It is small to dip in iodophor disinfection with cotton ball with ruler and the quasi- excision skin ranges of marking pen label back of mice skin 1cm × 1cm for plate
Mouse skin of back 3 times, 75% cotton ball soaked in alcohol takes off iodine；
(3) full thickness skin is cut off in the prior mark of back of mice with eye scissors, the wound of 1cm × 1cm skin full-thickness defects is made
Face；
(4) ruler is placed on beside wound and makees scale, digital camera record.Camera angle vertical is carried on the back in mouse when taking pictures wound and
Scale；
(5) keep the surface of a wound wet during the experiment, PBS buffer solution or physiological saline can be used for wet wound；
(6) square materials will be got out in advance to spread on the surface of a wound, outer patch operation patch towel, only towel is pasted in outer patch operation to control group, point
Cage raising；
(7) postoperative 1st day, remove operation patch towel.
Wounds in mice healing DATA REASONING
Wound healing rate calculation basis: Wound healing rate=(remaining surface of a wound area after original surface of a wound area-N days wounds)/former
Firstly appear face area × 100%.
Original surface of a wound area and each time of measuring point surface of a wound are measured with IPP6.0 software, method particularly includes: starting IPP6.0
Software, is placed on the scale Set scale ruler of the ruler by the surface of a wound when shooting, then imports picture；Wound is selected to use software
AOI function；It measures wound area and uses " count size " function；Row scale bar converts to obtain practical surface of a wound area.
Interpretation of result
Influence of the inventor with regard to different materials to wound healing is studied, such as Figure 17, Figure 18, * P < 0.01, n=3.
The result shows that postoperative 3rd day of bionical bilayer dressing group and the 7th day Wound healing rate are obviously accelerated；The 3rd day after wound, invention human hair
Existing bionical double-deck dressing group (healing rate 63.22%) healing rate be significantly faster than that gelatin foam group (healing rate 21.32%) and
Control group (healing rate 12.18%), significant difference.Control group wound inflammation is heavier and dries, and wound healing is poor, and wound contraction is not
Obviously；SIS film group has a certain amount of secretion in wound surface, uses the bionical double-deck dressing, freezing gel and gelfoam
The surface of a wound for the treatment of, wound clean, no exudation, wherein bionical bilayer dressing group wound healing is more preferable.
As shown in figure 18, postoperative 7th day bionical double-deck dressing group (SIS bilayer) surface of a wound has more than 90% healing, empty
White control group is lower than 50%, and comparing difference is statistically significant.The healing rate of freezing gel group and SIS film group wound is respectively
87.64%, 79.83%, it is not statistically significant with the bionical double-deck dressing group comparing difference；Gelatine sponge wound healing rate
67.27%, the significant difference compared with the bionical double-deck dressing, bionical bilayer dressing group wound healing is substantially speeded up.
Claims (9)
1. a kind of bionical double-deck dressing, the gel layer including film layer and crosslinking in film layer, the film layer and the common structure of gel layer
At the bionical double-deck dressing, it is characterised in that: the film layer is de- multicellular animal submucous layer of small intestine, and the gel layer is with animal
Submucous layer of small intestine is freezing gel made from raw material.
2. the bionical double-deck dressing according to claim 1, it is characterised in that: the animal mucous membrane of small intestine be pig, ox, sheep,
One of horse or monkey mucous membrane of small intestine.
3. the bionical double-deck dressing according to claim 2, it is characterised in that: be distributed with and be mutually communicated on the gel layer
Micropore, and the average pore size of micropore is 50-400 μm.
4. the bionical double-deck dressing according to claim 2 or 3, it is characterised in that: the thicknesses of layers is 80-100 μm, institute
Stating gel layer thicknesses is 180-220 μm.
5. the preparation method of the bionical double-deck dressing according to any one of claims 1-4, it is characterised in that: small including de- cell
Prepared by intestinal submucosa preparation, de- cell submucous layer of small intestine powder, prepared by de- cell submucous layer of small intestine solution and bionical double
Layer dressing preparation step, specifically:
Step 1, it takes off the preparation of cell submucous layer of small intestine: taking fresh chitterlings, first remove placenta percreta and muscle layer；Then it places it in
It is impregnated 6-24 hours in volume ratio=1.2-2:1 methanol and chloroform mixed liquor, deionized water rinsing；Then by the pig after rinsing
Small intestine is placed in 0.025-0.1% pancreatin and the mixed system of 0.05-0.1% ethylenediamine tetra-acetic acid that digest 8-15 at room temperature small
When, water cleaning；It is small that chitterlings after water is cleaned again are placed in oscillation 6-24 in the physiological saline containing 0.5% lauryl sodium sulfate
When, obtain submucous layer of small intestine；
Step 2, the preparation of cell submucous layer of small intestine powder is taken off: after gained is taken off cell submucous layer of small intestine acetic acid soaked overnight
It crushes, is placed in after being digested in pepsin and filters, gained filtrate takes its precipitating after 1-4 times is saltoutd centrifugation；Then by gained
Precipitating is dissolved in acetic acid, and dialysis obtained dialyzate after 15-72 hours；Dialyzate is lyophilized again, is obtained under spongy mucous membrane of small intestine
Layer obtains de- cell submucous layer of small intestine powder finally by the spongy de- cell submucous layer of small intestine grind into powder of gained；
Step 3, it takes off the preparation of cell submucous layer of small intestine solution: gained is taken off into cell submucous layer of small intestine powder by final concentration 8-
20mg/ml is dissolved in 2- (N- morpholine) ethanesulfonic acid or phosphate of pH=3-7, is obtained after mixing evenly under the conditions of 3-8 DEG C
De- cell submucous layer of small intestine solution；
Step 4, the bionical double-deck dressing preparation: de- cell submucous layer of small intestine is placed in buffer after impregnating 10-24 hours and is put down
It is laid in culture dish, suck dry moisture；Then de- cell submucous layer of small intestine solution is mixed with crosslinked fluid, is mixed after stirring
System；De- cell submucous layer of small intestine surface is added in gained mixed system again, is placed in subzero 20 DEG C to subzero 180 DEG C of ring
It is freezed in border 15-30 hours, the bionical double-deck dressing is obtained after dissolving at room temperature.
6. the preparation method of the bionical double-deck dressing according to claim 5, it is characterised in that: the buffer is that hydrochloric acid is molten
Liquid, the crosslinked fluid are glutaraldehyde or tea polyphenols and phosphatic mixed solution.
7. the preparation method of the bionical double-deck dressing according to claim 6, it is characterised in that: the final concentration of the glutaraldehyde
For 0.075-1%.
8. the preparation method of the bionical double-deck dressing according to claim 6, it is characterised in that: the final concentration of the tea polyphenols
For 0.5-4g/L.
9. the preparation method of the bionical double-deck dressing according to claim 5, it is characterised in that: the buffer is 2- (N-
Morpholine) ethanesulfonic acid, the crosslinked fluid is 1- ethyl -3- (3- dimethylaminopropyl) carbodiimide hydrochloride solution or hydroxysuccinimidyl
Imide solution.
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| WO2022229653A1 (en) * | 2021-04-30 | 2022-11-03 | University Of Nottingham | Decellularized tissue hydrogels |
| CN115869453A (en) * | 2021-09-26 | 2023-03-31 | 中国科学院理化技术研究所 | A double-layer antibacterial dressing loaded with antibacterial molecules, its preparation and application |
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