CN114191613A - Biological cornea prepared from swimming bladder as well as preparation method and application of biological cornea - Google Patents
Biological cornea prepared from swimming bladder as well as preparation method and application of biological cornea Download PDFInfo
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- 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
- A61L27/3683—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
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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
- A61L27/3683—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
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- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/16—Materials or treatment for tissue regeneration for reconstruction of eye parts, e.g. intraocular lens, cornea
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- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
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- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Abstract
The invention discloses a biological cornea prepared from swim bladder and a preparation method and application thereof. The obtained biological cornea is very close to a natural cornea in components, has excellent optical characteristics and excellent mechanical properties, shows excellent biocompatibility and no cytotoxicity by culturing cells in vitro, can normally adhere and grow on the surface of the material to form a compact cell layer, and can be used as a medical material in corneal transplantation and corneal lamellar repair. The invention has wide raw material source, low production cost, convenient storage and use and can meet the requirement of a large number of carrier supports of the tissue engineering cornea.
Description
Technical Field
The invention relates to the field of medical materials in medical science and bioengineering, in particular to a biological cornea prepared from swim bladder and a preparation method and application thereof.
Background
Keratopathy is one of the major eye diseases worldwide and is also an important blinding disease. The incidence of disease has increased year by year in many countries and has become the second most largely blind eye disease in some developing countries in recent years. The pathological changes of the corneal stroma are usually the destruction of the arrangement sequence of the stroma collagen lamellae. The healing time of the corneal stroma is months or even years, and the healing wound can generate scars to influence the corneal definition, so that the visual effect is usually caused by stroma pathological changes. Corneal diseases of stroma layer include keratoconus with thinned central cornea and forward protrusion; most of the diseases are characterized by autosomal dominant inheritance, generally symmetrical pathogenesis of eyes and stroma malnutrition which is unrelated to primary corneal inflammation and systemic diseases; there are infectious keratopathy caused by bacteria, fungi, viruses, mycoplasma, etc. Severe corneal patients require vision to be obtained by transplantation of a healthy cornea instead of a damaged cornea. The high incidence and blindness rates have led to an increasing demand for corneal donor numbers year by year, and an effective eye vault system and an adequate number of corneal surgeons are the guarantee of success in corneal transplants. However, in China, the eye bank infrastructure is underdeveloped, the eye bank standard is not available or is not complete, and the cornea donor is extremely short, so that a large number of patients cannot be treated in time.
With the continuous development of cell biology, molecular biology and materials science, the development of tissue engineering technology is rapid, and the application of tissue engineering technology in vitro reconstruction of artificial corneas becomes a research hotspot in recent years. The carrier support materials for constructing the tissue engineering artificial cornea at present mainly comprise allogenic cornea (human), xenogenic acellular cornea (pig), natural biological materials and artificial synthetic materials, but all have certain defects and shortcomings: the xenogenic acellular corneal stroma such as porcine cornea can destroy the original structure and components of the stroma in the process of preparing acellular stroma, influence the transparency and mechanical strength of the stroma, and can cause inflammatory reaction and transparency reduction after transplantation; the degradation rate of the scaffold made of natural biological materials such as collagen, chitosan, fibrin and the like is too fast, and the mechanical strength is not good enough; artificially synthesized materials sometimes suffer from poor biocompatibility, and their degradation products may have some toxic effects on organisms. Therefore, the preparation of the scaffold material with stable physicochemical property, high transparency and good mechanical property, and good biocompatibility with seed cells is still a difficult point to overcome in the field of tissue engineering cornea.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a biological cornea which has similar components and structures as the natural cornea, excellent optical characteristics and mechanical properties and good biocompatibility and is prepared by swim bladders;
the second purpose of the invention is to provide a method for preparing biological cornea by using swim bladder;
the third purpose of the invention is to provide the application of the biological cornea prepared by the swim bladder in the field of tissue engineering cornea.
The purpose of the invention is realized by the following technical scheme: a method for preparing biological cornea with swim bladder comprises removing impurities from fresh swim bladder, defatting, desugarizing, freezing and thawing, adding cell-free solution to obtain cell-free matrix of swim bladder, and reconstructing collagen fiber with alcohol-water mixed solution containing light transmittance regulator to obtain artificial biological cornea.
Further, the light transmittance regulator is at least one of formaldehyde, glutaraldehyde, 1, 4-butanediol diglycidyl ether, propylene glycol diglycidyl ether, ethylene glycol diglycidyl ether, glycerol triglycidyl ether, 3- (perfluoro-n-octyl) -1, 2-epoxypropane, glyoxylic acid, hexamethylene dicyanate, azidodiphenylphosphine, curcumin, procyanidine, tea polyphenol and epigallocatechin gallate.
Further, the content of the light transmittance regulator in the alcohol-water mixed solution containing the light transmittance regulator is 0.5-10% by mass, the content of the alcohol in the alcohol-water mixed solution is 5-95%, and the alcohol is at least one of methanol, ethanol, ethylene glycol and glycerol.
Further, the swim bladder is selected from any one of carp, grass carp, crucian carp, silver carp, black carp, bighead carp, bream, yellow croaker, sturgeon or spanish mackerel.
Further, the degreasing method comprises the following steps: adding degreasing liquid into the fresh swimming bladders after impurity removal, controlling the speed of a shaking table to be 50-150 r/min, and carrying out degreasing treatment for 0.5-2 h, wherein the degreasing liquid is a mixed solution of a liquid A and a liquid B, the liquid A is at least one of dichloromethane, trichloromethane, carbon tetrachloride, trichloroethylene and tetrachloroethylene, the liquid B is at least one of methanol, ethanol or glycerol, and the volume ratio of the liquid A to the liquid B is 0.1-10: 1.
Further, the method for the desugaring treatment comprises the following steps: adding 1-10% by mass of sodium chloride solution into the degreased swimming bladder, controlling the speed of a shaking table to be 50-150 r/min, and carrying out desugarization treatment for 0.5-2 h.
Further, after freezing and thawing the swimming bladders, firstly adding the cell removal liquid I, placing the swimming bladders in a shaking table for 2-72 h, then adding the cell removal liquid II, and carrying out shaking table treatment for 2-72 h, wherein the speed of the shaking table is controlled to be 100-200 r/min; the solute of the cell removal liquid I is at least one of sodium dodecyl sulfate, sodium deoxycholate, triton X-100, triton X-200, 3- [ (3-cholesteryl aminopropyl) dimethylamino ] -1-propanesulfonic acid, hydrochloric acid, peracetic acid, ethylene diamine tetraacetic acid, ethylene glycol tetraacetic acid, sodium hydroxide and glycerol, and the solute of the cell removal liquid II is at least one of nuclease, trypsin, neutral protease, phospholipase A2, thermolysin, alpha-galactosidase and lipase.
Furthermore, the cell removal solution I is 0.5-10% in mass percentage, and the cell removal solution II is 0.5-100 mug/ml in concentration.
The artificial biological cornea prepared by the method.
The artificial biological cornea is applied as a medical material in corneal transplantation and corneal lamella repair.
Swim bladder is a membranous organ existing in most bony fishes, and the position of the swim bladder in water is continuously changed by continuously contracting and expanding to adjust the air content. The swimming bladder mainly comprises type I collagen, wherein the extracted collagen has good histocompatibility, low antigenicity and degradability, and good physicochemical properties of promoting cell adhesion, growth and proliferation and the like. Swim bladder source biomembrane materials have been researched to have excellent anti-calcification capability and mechanical property, and can be applied to the repair field of dura mater, blood vessels, skin and the like. According to the component research of the existing natural biological material swim bladder, aiming at the previous research difficulty of the tissue engineering cornea, the swim bladder is selected as the raw material. The main component of the swimming bladder is type I collagen, but the light transmittance of the swimming bladder is not good, and the untreated swimming bladder can not meet the light transmittance requirement of a tissue engineering cornea completely. According to the invention, a proper light transmittance regulator is selected, and then the light transmittance of the material is greatly improved through the selection of a light transmittance regulator solvent, the mechanical property of the material after inspection treatment is enhanced, the biocompatibility is good, the stability is strong, and the material is hopefully and successfully applied to the field of clinical restoration of tissue engineering cornea.
The invention has the following advantages:
(1) the swim bladder is used as a material for preparing the biological cornea, the obtained biological cornea is very close to a natural cornea in components, and has excellent optical characteristics and excellent mechanical properties, through detection, the light transmittance of the swim bladder biological cornea prepared by the method is 80-95% in a visible light wavelength range, the light transmittance of the biological cornea prepared by the swim bladder is effectively improved, the light transmittance of the biological cornea is very close to that of a contact lens, the aim of improving or recovering vision can be achieved, the biocompatibility is good, and the requirement of a tissue engineering cornea on a scaffold material is met.
(2) The preparation method of the invention avoids the phenomena that the fibers in the matrix layer swell and are disorderly arranged, thereby affecting the light transmittance and the mechanical strength of the material. The cornea cell can be normally adhered and grown on the surface of the material to form a compact cell layer.
(3) The biological cornea prepared by the swim bladder is a potential excellent cornea substitute, can be used for various corneal lamellar transplantation indications such as keratitis, corneal dystrophy, corneal degeneration and the like, and can be directly applied to clinical transplantation and treatment of various keratoses; and the raw material source is wide, the production cost is low, the storage and the use are convenient, and the requirements of a large number of carrier supports of the tissue engineering cornea can be met.
Drawings
FIG. 1 is a scanning electron microscope cross-sectional photograph of a layered structure of swim bladder material.
FIG. 2 is a comparison of before and after swim bladder treatment by the method of the present invention.
FIG. 3 is a graph of the transmittance of air bladder before and after treatment by the method of the present invention in the visible wavelength range.
FIG. 4 is a graph showing the cell viability of L929 cells cultured on biological corneas prepared by the method of the present invention.
FIG. 5 is an optical microscope photograph of rabbit corneal stromal cells cultured on a biological cornea prepared by the method of the present invention.
Detailed Description
The invention is further described with reference to the following figures and examples, without limiting the scope of the invention to the following:
example 1:
a method for preparing biological cornea with swim bladder comprises removing impurities from swim bladder of fresh Spanish bladder, defatting, desugarizing, freezing and thawing, adding cell-free solution to obtain acellular matrix of swim bladder, and reconstructing the acellular matrix with alcohol-water mixed solution containing light transmittance regulator to obtain artificial biological cornea.
The preparation method comprises the following steps:
removing impurities from fresh mackerel swim bladder, adding a mixed solution of dichloromethane and ethanol, wherein the volume ratio of dichloromethane to ethanol is 0.1:1, controlling the speed of a shaking table to be 50 r/min, and carrying out degreasing treatment for 0.5 h;
adding 1% sodium chloride solution in mass percent into the degreased swimming bladder, controlling the shaking table speed at 50 r/min, and performing desugarization treatment for 0.5 h;
after desugarizing treatment, freezing and thawing swim bladders, adding a lauryl sodium sulfate solution with the mass percent content of 0.5%, placing in a shaking table for 2h, then adding a trypsin solution with the concentration of 0.5 mu g/ml, and carrying out shaking table treatment for 2h, and controlling the speed of the shaking table to be 100 r/min to obtain a acellular matrix of the swim bladders;
adding an alcohol-water mixed solution containing a light transmittance regulator glutaraldehyde into the acellular matrix to carry out collagen fiber reconstruction treatment, wherein the light transmittance regulator in the methanol-water mixed solution containing the light transmittance regulator accounts for 0.5% by mass, and the alcohol in the alcohol-water mixed solution accounts for 5% by mass;
placing the processed air bladder in the cell culture pore plate with the cover opened, performing double-sided ultraviolet sterilization for 30min respectively, and performing ultraviolet irradiation with energy of 100 mj/cm2And obtaining the artificial biological cornea.
Example 2:
a method for preparing biological cornea with swim bladder comprises removing impurities from swim bladder of fresh silver carp, defatting, desugarizing, freezing and thawing, adding cell-free solution to obtain cell-free matrix of swim bladder, and reconstructing the cell-free matrix with alcohol-water mixed solution containing light transmittance regulator to obtain artificial biological cornea.
The preparation method comprises the following steps:
removing impurities from fresh air bladders of chubs, adding degreasing liquid, wherein the degreasing liquid is a mixed solution of a liquid A and a liquid B, the liquid A is a mixed solution of carbon tetrachloride and trichloroethylene, the liquid B is a mixed solution of methanol and ethanol, the volume ratio of the liquid A to the liquid B is 10:1, the speed of a shaking table is controlled to be 150 r/min, and degreasing treatment is carried out for 2 hours;
adding 10% sodium chloride solution into degreased air bladder, controlling shaking table speed at 150 r/min, and desugarizing for 2 h;
after desugarizing treatment, freezing and thawing the swimming bladder, adding the cell removal solution I, placing in a shaking table for 72h, then adding the cell removal solution II, and carrying out shaking table treatment for 72h, wherein the speed of the shaking table is controlled to be 200 r/min; the cell removal solution I is a mixed solution of sodium deoxycholate, Triton X-100 and 3- [ (3-cholesteryl aminopropyl) dimethylamino ] -1-propanesulfonic acid, the mass percentage content of the cell removal solution I is 10%, the cell removal solution II is a mixed solution of neutral protease, phospholipase A2 and thermolysin, and the concentration of the cell removal solution II is 100 mu g/ml, so that the cell removal matrix of the swimming bladder is obtained;
adding an alcohol-water mixed solution containing a light transmittance regulator into the acellular matrix to carry out collagen fiber reconstruction treatment, wherein the light transmittance regulator is ethylene glycol diglycidyl ether, glycerol triglycidyl ether, 3- (perfluoro-n-octyl) -1, 2-epoxypropane and tea polyphenol, the mass percentage content of the light transmittance regulator in the alcohol-water mixed solution containing the light transmittance regulator is 10%, the content of alcohol in the alcohol-water mixed solution is 95%, and the alcohol is a mixed solution of ethanol, ethylene glycol and glycerol;
placing the processed swimming bladder in a cell culture pore plate with an open cover, performing double-sided ultraviolet sterilization for 60min respectively, and performing ultraviolet irradiation with energy of 300 mj/cm2 to obtain the artificial biological cornea.
Example 3:
a method for preparing biological cornea with swim bladder comprises removing impurities from swim bladder of fresh Cyprinus Carpio, defatting, desugarizing, freezing and thawing, adding cell-free solution to obtain cell-free matrix of swim bladder, and reconstructing the cell-free matrix with alcohol-water mixed solution containing light transmittance regulator to obtain artificial biological cornea.
The preparation method comprises the following steps:
removing impurities from fresh air bladders of carps, adding degreasing liquid, wherein the solution A is a mixed solution of a solution A and a solution B, the solution A is a mixed solution of dichloromethane, trichloroethylene and tetrachloroethylene, the solution B is a mixed solution of methanol, ethanol and glycerol, the volume ratio of the solution A to the solution B is 3:1, the speed of a shaking table is controlled to be 80 r/min, and degreasing treatment is carried out for 1 h;
adding 3% sodium chloride solution by mass into degreased air bladder, controlling shaking table speed at 85 r/min, and desugaring for 1 h;
after desugarizing treatment, freezing and thawing the swimming bladder, adding the cell removal solution I, placing in a shaking table for 22h, then adding the cell removal solution II, and carrying out shaking table treatment for 30h, wherein the speed of the shaking table is controlled to be 180 r/min; the cell removal solution I is a mixed solution of Triton X-200, 3- [ (3-cholesteryl aminopropyl) dimethylamino ] -1-propanesulfonic acid, hydrochloric acid and peroxyacetic acid, the mass percentage content of the cell removal solution I is 3.5%, the cell removal solution II is a mixed solution of nuclease, trypsin, neutral protease, phospholipase A2, thermolysin, alpha-galactosidase and lipase, and the concentration of the cell removal solution II is 28 mug/ml, so that the cell removal matrix of the swimming bladder is obtained;
adding an alcohol-water mixed solution containing a light transmittance regulator into the acellular matrix to carry out collagen fiber reconstruction treatment, wherein the light transmittance regulator is propylene glycol diglycidyl ether, ethylene glycol diglycidyl ether, glycerol triglycidyl ether and diphenylphosphoryl azide, the mass percentage of the light transmittance regulator in the alcohol-water mixed solution containing the light transmittance regulator is 3%, and the alcohol in the alcohol-water mixed solution is glycerol with the content of 28%;
placing the processed air bladder in the cell culture pore plate with the cover opened, performing double-sided ultraviolet sterilization for 40min respectively, and performing ultraviolet irradiation with energy of 180 mj/cm2And obtaining the artificial biological cornea.
Example 4:
a method for preparing biological cornea with swim bladder comprises removing impurities from fresh swim bladder of Aristichthys nobilis, defatting, desugarizing, freezing and thawing, adding cell-free solution to obtain cell-free matrix of swim bladder, and reconstructing the cell-free matrix with alcohol-water mixed solution containing light transmittance regulator to obtain artificial biological cornea.
The preparation method comprises the following steps:
removing impurities from fresh bighead swimming bladders, adding degreasing liquid, wherein the degreasing liquid is mixed solution of liquid A and liquid B, the liquid A is mixed solution of dichloromethane, trichloromethane, carbon tetrachloride and trichloroethylene, the liquid B is methanol, the volume ratio of the liquid A to the liquid B is 8:1, the speed of a shaking table is controlled to be 120 r/min, and degreasing treatment is carried out for 1.5 hours;
adding 7% sodium chloride solution into degreased air bladder, controlling shaking table speed at 125 r/min, and desugaring for 1.5 h;
after desugarizing treatment, freezing and thawing the swimming bladder, adding the cell removal solution I, placing in a shaking table for 50h, then adding the cell removal solution II, and carrying out shaking table treatment for 60h, wherein the speed of the shaking table is controlled to be 180 r/min; the cell removal solution I is a mixed solution of sodium dodecyl sulfate, sodium deoxycholate, triton X-100, triton X-200, 3- [ (3-cholesteryl aminopropyl) dimethylamino ] -1-propanesulfonic acid, hydrochloric acid, peracetic acid, ethylene diamine tetraacetic acid, ethylene glycol tetraacetic acid, sodium hydroxide and glycerol, the mass percentage content of the cell removal solution I is 8%, the cell removal solution II is a mixed solution of nuclease, trypsin, neutral protease and thermolysin, and the concentration of the cell removal solution II is 80 mu g/ml, so that the cell removal matrix of the swimming bladder is obtained;
adding an alcohol-water mixed solution containing a light transmittance regulator into the acellular matrix to carry out collagen fiber reconstruction treatment, wherein the light transmittance regulator is formaldehyde, glutaraldehyde, 1, 4-butanediol diglycidyl ether, propylene glycol diglycidyl ether, ethylene glycol diglycidyl ether, glycerol triglycidyl ether, 3- (perfluoro-n-octyl) -1, 2-epoxypropane, glyoxylic acid, hexamethylene dicyanate, azidodiphenylphosphine, curcumin, procyanidine, tea polyphenol and epigallocatechin gallate, the mass percentage of the light transmittance regulator in the alcohol-water mixed solution containing the light transmittance regulator is 7.8%, the alcohol content in the alcohol-water mixed solution is 82%, and the alcohol is a mixture of methanol, ethanol and ethylene glycol;
placing the processed air bladder in the cell culture pore plate with the cover opened, performing double-sided ultraviolet sterilization for 48min respectively, and performing ultraviolet irradiation with energy of 260 mj/cm2And obtaining the artificial biological cornea.
The swim bladder can be swim bladder of carp, grass carp, crucian carp, silver carp, black carp, bighead carp, bream, yellow croaker, sturgeon or spanish mackerel, and the light transmittance regulator is one or more of formaldehyde, glutaraldehyde, 1, 4-butanediol diglycidyl ether, propylene glycol diglycidyl ether, ethylene glycol diglycidyl ether, glycerol triglycidyl ether, 3- (perfluoro-n-octyl) -1, 2-epoxypropane, glyoxylic acid, hexamethylene dicyanate, azido diphenyl phosphate, curcumin, procyanidine, tea polyphenol and epigallocatechin gallate; the liquid A in the degreasing solution is at least one of dichloromethane, trichloromethane, carbon tetrachloride, trichloroethylene and tetrachloroethylene, and the liquid B is at least one of methanol, ethanol or glycerol; the solute of the cell removal liquid I is at least one of sodium dodecyl sulfate, sodium deoxycholate, triton X-100, triton X-200, 3- [ (3-cholesteryl aminopropyl) dimethylamino ] -1-propanesulfonic acid, hydrochloric acid, peroxyacetic acid, ethylene diamine tetraacetic acid, ethylene glycol tetraacetic acid, sodium hydroxide and glycerol, and the solute of the cell removal liquid II is at least one of nuclease, trypsin, neutral protease, phospholipase A2, thermolysin, alpha-galactosidase and lipase. The best combination of the embodiments is only listed in the specific embodiments of the invention, and other combinations and contents which are not listed only need to be replaced or changed equivalently, so that the combinations and contents are not listed any more.
The following experiments illustrate the beneficial effects of the present invention:
experimental example 1:
fresh air bladders of carps are taken, the cross section structure of the air bladders is characterized, the air bladders are subjected to freeze-drying treatment, then trimmed into a proper size and placed in a sample pile, a vacuum spraying instrument is placed for metal spraying treatment, and then scanning electron microscope observation is carried out, so that the section view of the laminar structure of the air bladder material in the experimental example under a scanning electron microscope is obtained, and is shown in figure 1.
Experimental example 2:
the light transmittance of a fresh carp swim bladder sample and a Haichang contact lens before and after treatment is measured in a visible wavelength range (400-.
The treatment method specifically comprises the following steps:
cleaning fresh air bladders of the carps in water, removing surface impurities, and cutting;
carrying out degreasing and sugar removal treatment: removing impurities from fresh air bladders of carps, adding a mixed solution of carbon tetrachloride and ethanol, controlling the volume ratio of the carbon tetrachloride to the ethanol to be 3:1, controlling the speed of a shaking table to be 80 r/min, and carrying out degreasing treatment for 1 h; adding 3% sodium chloride solution by mass into degreased air bladder, controlling shaking table speed at 85 r/min, and desugaring for 1 h;
degreasing, desugarizing, performing freeze thawing for multiple times, placing the freeze-thawed swimming bladders in a 1% ethylenediaminetetraacetic acid solution, and treating at normal temperature for 24h, wherein the shaking table speed is controlled to be 120 r/min;
placing the mixture in 25 microgram/ml alpha-galactosidase solution for shaking treatment for 6 hours, and controlling the speed of a shaking table to be 120 r/min; then rinsing the mixture for 12 hours in sterile PBS solution;
placing the acellular matrix in 0.5% ethylene glycol diglycidyl ether methanol solution for treatment for 12h to obtain an acellular matrix with improved light transmittance of swimming bladders;
soaking the obtained acellular matrix with improved light transmittance in pure water, and ultrasonically cleaning for 1-2h at controlled temperature; then ultraviolet sterilization is carried out, wherein the ultraviolet sterilization is that swim bladders are placed in a cell culture pore plate with an opened cover, the double-sided ultraviolet sterilization is carried out for 40min respectively, and the ultraviolet irradiation energy is 220 mj/cm2Soaking in 75% alcohol overnight, cleaning and storing.
Experimental example 3:
after sterilizing the swimming bladder of the carp treated in the experimental example 2, performing a CCK-8 cytotoxicity detection experiment, inoculating L929 cells with proper concentration into a 96-well plate, culturing the fish through a complete culture medium and a leaching solution, 1/3/5day, fixing a point, adding a proper amount of CCK-8 reagent into the well plate to detect the absorbance value, and comparing the absorbance values, wherein the obtained material has good biocompatibility as shown in figure 4; under light microscopy, rabbit corneal stromal cells on the material treated by the method of the invention attached well, as shown in FIG. 5.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can substitute or change the technical solution of the present invention and the inventive concept within the technical scope of the present invention.
Claims (10)
1. A method for preparing a biological cornea by using swimming bladders is characterized in that fresh swimming bladders are subjected to degreasing, desugarizing and freeze-thawing treatment after being subjected to impurity removal, then cell-free liquid is added to obtain a cell-free matrix of the swimming bladders, and the cell-free matrix is subjected to collagen fiber reconstruction treatment by using alcohol-water mixed liquid containing a light transmittance regulator to obtain the artificial biological cornea.
2. The method for preparing biological cornea with swimming bladder as claimed in claim 1, wherein the light transmittance regulator is at least one of formaldehyde, glutaraldehyde, 1, 4-butanediol diglycidyl ether, propylene glycol diglycidyl ether, ethylene glycol diglycidyl ether, glycerol triglycidyl ether, 3- (perfluoro-n-octyl) -1, 2-epoxypropane, glyoxylic acid, hexamethylene dicyanate, azidodiphenylphosphine, curcumin, procyanidins, tea polyphenols and epigallocatechin gallate.
3. The method for preparing a biological cornea with swim bladder as claimed in claim 1, wherein the content of the light transmittance regulator in the alcohol-water mixture containing the light transmittance regulator is 0.5-10% by mass, the content of the alcohol in the alcohol-water mixture is 5-95% by mass, and the alcohol is at least one of methanol, ethanol, ethylene glycol and glycerol.
4. The method for preparing the biological cornea by using swim bladders as claimed in claim 1, wherein the swim bladders are selected from any one of carp, grass carp, crucian carp, silver carp, black carp, bighead carp, bream, yellow croaker, sturgeon or spanish mackerel.
5. The method for preparing a biological cornea with swim bladder as claimed in claim 1, wherein the degreasing method comprises: adding degreasing liquid into the fresh swimming bladders after impurity removal, controlling the speed of a shaking table to be 50-150 r/min, and carrying out degreasing treatment for 0.5-2 h, wherein the degreasing liquid is a mixed solution of a liquid A and a liquid B, the liquid A is at least one of dichloromethane, trichloromethane, carbon tetrachloride, trichloroethylene and tetrachloroethylene, the liquid B is at least one of methanol, ethanol or glycerol, and the volume ratio of the liquid A to the liquid B is 0.1-10: 1.
6. The method for preparing a biological cornea with swim bladder as claimed in claim 1, wherein the desugaring treatment method comprises: adding 1-10% by mass of sodium chloride solution into the degreased swimming bladder, controlling the speed of a shaking table to be 50-150 r/min, and carrying out desugarization treatment for 0.5-2 h.
7. The method for preparing a biological cornea by using swim bladders as claimed in claim 1, wherein after the swim bladders are frozen and thawed, firstly adding the cell removal solution I and placing the cell removal solution I in a shaking table for 2-72 h, then adding the cell removal solution II and carrying out shaking table treatment for 2-72 h, and controlling the speed of the shaking table to be 100-200 r/min; the solute of the cell removal liquid I is at least one of sodium dodecyl sulfate, sodium deoxycholate, triton X-100, triton X-200, 3- [ (3-cholesteryl aminopropyl) dimethylamino ] -1-propanesulfonic acid, hydrochloric acid, peracetic acid, ethylene diamine tetraacetic acid, ethylene glycol tetraacetic acid, sodium hydroxide and glycerol, and the solute of the cell removal liquid II is at least one of nuclease, trypsin, neutral protease, phospholipase A2, thermolysin, alpha-galactosidase and lipase.
8. The method for preparing a biological cornea with swim bladder as claimed in claim 7, wherein the cell-free fluid I is 0.5-10% by weight, and the cell-free fluid II is 0.5-100 μ g/ml.
9. An artificial biological cornea made according to the method of any one of claims 1-8.
10. The artificial biocornea of claim 9 for use as a medical material in corneal transplantation and corneal lamellar repair.
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