CN106039403B - Biologically active corneal restoration plants the preparation method of piece and corneal restoration plants piece - Google Patents
Biologically active corneal restoration plants the preparation method of piece and corneal restoration plants piece Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/24—Collagen
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- 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
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- C08L89/00—Compositions of proteins; Compositions of derivatives thereof
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/18—Modification of implant surfaces in order to improve biocompatibility, cell growth, fixation of biomolecules, e.g. plasma treatment
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- 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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Abstract
The preparation method of piece is planted the invention discloses a kind of biologically active corneal restoration and corneal restoration plants piece, solves the technical issues of corneal donor lacks.It include: to prepare collagen swelling solution；Based on the collagen swelling solution, collagem membrane is prepared；Aseptically, increase surface-functionalized modified material in the collagen film surface in a manner of 3D printing or coating, plant piece to form biologically active corneal restoration.It is that fine particle is prepared in material that the surface-functionalized modified material, which is by the corneal stroma containing competent cell of the self removal lesion portion of sufferer, biology, the biological stability of high-intensitive collagem membrane can be improved, improve the physiological function of cornea, there is the good transparency and moisture content after making modified collagem membrane implantation sufferer glasses, the transparency can clinically be restored faster, have good bio-compatible performance, provides good bracket for the keratocyte culture of corneal transplantation receptor.
Description
Technical field
The invention belongs to tissue engineering technique fields, specifically, being to be related to a kind of biologically active corneal restoration
It plants the preparation method of piece and plants piece using biologically active corneal restoration prepared by the preparation method.
Background technique
Cornea be with eyeball antetheca, be a kind of hyaline tissue, it is main to exercise barrier and dioptric function.
Since cornea is directly contacted with the external world, it is highly prone to mechanical trauma, thermal burn, soda acid erosion etc., so as to cause angle
The generation of membrane disease, serious person even blind.Present corneal blindness has been deputy diseases causing blindness in the whole world, and the whole world is annual big
There are about up to ten million people to blind because of disease of cornea, and 80% can be cast off illiteracy by corneal graft among these.
But corneal blindness can not but be cast off illiteracy in default of corneal donor by corneal graft.
Summary of the invention
The preparation method of piece is planted this application provides a kind of biologically active corneal restoration and corneal restoration plants piece, solution
The technical issues of certainly corneal donor lacks.
In order to solve the above technical problems, the application is achieved using following technical scheme:
It is proposed that a kind of biologically active corneal restoration plants the preparation method of piece, which comprises the steps of:
Prepare collagen swelling solution；Based on the collagen swelling solution, collagem membrane is prepared；Aseptically, with the side of 3D printing or coating
Formula increases surface-functionalized modified material in the collagen film surface, plants piece to form biologically active corneal restoration.
It is further, described to prepare collagen swelling solution, specifically: it is extracted in animal knot hoof tissue with protease digestion method
Collagen；The collagen of extraction is swollen using the malonic acid solution of 0.3%-0.5%, and mixes and is centrifuged off impurity, is obtained
To collagen swelling solution.
It is further, described to extract collagen with protease digestion method in animal knot hoof tissue, specifically: described
Protein enzyme solution is added by weight in animal knot hoof tissue；Stirring is placed on the proteolysis that setting time is carried out in isoperibol
It reacts, during which timing agitation；The enzymolysis reaction after setting time；The animal knot hoof tissue of enzymolysis reaction is used into steaming
Distilled water rinses and dries to obtain collagen.
It is further, described to prepare collagem membrane, specifically: the collagen swelling solution is saltoutd by being saturated inorganic salt solution
Concentration, obtains collagen aaerosol solution；The collagen aaerosol solution is subjected to centrifugation and precipitation and separation, obtains collagen gel；By institute
After stating collagen gel merging mold, cryogenic freezing is freeze-dried；The collagen gel of freeze-drying is placed in two polytetrafluoros
It is flattened with smooth object as dense collagenous layer between ethylene plate；The dense collagenous layer flattened is carried out at crosslinking
Reason obtains collagem membrane.
Further, the preparation step of the surface-functionalized modified material includes: the removal lesion that sufferer is self
The corneal stroma containing competent cell divided aseptically shreds in culture dish, is transferred in the glassware disinfected,
Increase clostridiopetidase A；After the corneal stroma for increasing the chopping of clostridiopetidase A is sufficiently shredded, it is prepared into micro-granulating device
Uniform fine particle；After fine particle centrifugation, collecting, biological fibrin glue is added, forms described surface-functionalized change
Property substance.
Further, the protease is the ficin that concentration is 0.01%-0.5%.
Further, crosslinker solution used in the crosslinking Treatment is glutaraldehyde solution, EDC solution, glutamine turn
Adnosine deaminase solution or epoxy alkyl solution；The mass concentration of the crosslinker solution is 1.5%-25%.
Further, the fine particle diameter is 100-300 microns.
Further, the main component of the biological fibrin glue is fibrin, fibrin ferment and ionic calcium soln.
It proposes that a kind of biologically active corneal restoration plants piece, is planted according to the biologically active corneal restoration
The preparation method of piece is prepared.
Compared with prior art, the advantages of the application and good effect is: the biologically active angle that the application proposes
The preparation method of piece is planted in film reparation and corneal restoration is planted piece and beaten after being extracted collagen swelling solution and preparing collagem membrane using 3D
In the surface-functionalized modified material of collagen film surface increase, which is by sufferer for print or the mode of coating
The corneal stroma containing competent cell of self removal lesion portion is that fine particle is prepared in material, in collagen film surface
Function of surface is carried out to collagem membrane to be modified, can improve physics, the biological stability of high-intensitive collagem membrane, and there is higher water suction
Property, rehydration and water-retaining property, improve the physiological function of cornea, make to have after modified collagem membrane implantation sufferer glasses good
The transparency and moisture content can clinically restore the transparency faster, have good bio-compatible performance, be corneal transplantation
The keratocyte culture of receptor provides good bracket.In collagem membrane preparation process, by the step such as saltouing, drying and be crosslinked
Suddenly, collagen film-strength obtained be higher than traditional collagen film, after water suction can free bend and folding so that corneal restoration plant
Piece has good mechanics and mechanical performance.Thereby, it is possible to provide keratocyte growth substrate for cornea receptor to realize cornea weight
It builds, solves the technical issues of corneal donor shortage, the corneal restoration that corneal blindness patient can be provided based on this case is planted piece and passed through
Corneal graft is cast off illiteracy.
After the detailed description of the application embodiment is read in conjunction with the figure, other features and advantages of the application will become more
Add clear.
Detailed description of the invention
Fig. 1 is the method flow diagram that the biologically active corneal restoration that the application proposes plants piece.
Specific embodiment
The specific embodiment of the application is described in more detail with reference to the accompanying drawing.
The present invention is intended to provide a kind of biologically active corneal restoration plants the preparation method of piece, and use the system
The biologically active corneal restoration of Preparation Method preparation plants piece, and it is for corneal blindness patient canthus that the corneal restoration of preparation, which plants piece,
The timbering material that theca cell lives and reproduces, mechanical strength is higher than traditional collagen film, using autologous patient corneal stroma system
Standby fine particle carries out function of surface to collagen film and is modified, and can improve physics, the Biostatic of high-intensitive collagem membrane
Property, the physiological function of cornea is also improved, the transparency can faster be restored by making modified collagem membrane clinically.
Biologically active corneal restoration proposed by the present invention plants the preparation method of piece, as shown in Figure 1, including following step
It is rapid:
Step S11: collagen swelling solution is prepared.
Specifically, extracting collagen with protease digestion method in animal knot hoof tissue；Then by the collagen of extraction
Albumen is swollen using the malonic acid solution of 0.3%-0.5%, and is mixed and be centrifuged off impurity, is obtained collagen swelling solution, is put into 4 DEG C of rings
It is saved backup under border.
Step S12: it is based on collagen swelling solution, prepares collagem membrane.
Specifically, collagen swelling solution is saltoutd concentration by being saturated inorganic salt solution, collagen aaerosol solution is obtained；By collagen
Aaerosol solution carries out centrifugation and precipitation and separation, obtains collagen gel of the moisture content less than 10%；Collagen gel is placed in mold,
It is freeze-dried after cryogenic freezing；Just being placed between two polytetrafluoroethylenes by gel for freeze-drying is smooth with Kun etc.
Object is flattened as dense collagenous layer；Dense collagenous layer progress crosslinking Treatment is finally obtained into collagem membrane.
Wherein, the inorganic salts being saturated in inorganic salt solution can be sodium salt, sylvite, calcium salt or magnesium salts.
The crosslinker solution used in crosslinking Treatment is glutaraldehyde solution, EDC solution, glutamine transaminage solution or ring
Oxyalkyl solution；The mass concentration of crosslinker solution is 1.5%-25%.
Step S13: aseptically, increase function of surface in the collagen film surface in a manner of 3D printing or coating
Change modified material, plants piece to form biologically active corneal restoration.
The preparation step of the surface-functionalized modified material is as follows: by the thin containing activity of the self removal lesion portion of sufferer
The corneal stroma of born of the same parents aseptically shreds in culture dish, is transferred in the glassware disinfected, increases clostridiopetidase A；It will
Increase the chopping of clostridiopetidase A corneal stroma sufficiently shred after, uniform subtle is prepared into micro-granulating device
Grain；After fine particle centrifugation, collecting, biological fibrin glue is added, forms the surface-functionalized modified material.
Among these, fine particle diameter needs between 100 microns to 300 microns；The main component of biological fibrin glue is
Fibrin, fibrin ferment and ionic calcium soln etc..
Above-mentioned, biologically active corneal restoration proposed by the present invention is planted in the preparation method of piece, is being extracted collagen
After swelling solution prepares collagem membrane, increase surface-functionalized modifier in collagen film surface by the way of 3D printing or coating
Matter, it is material which, which is by the corneal stroma containing competent cell of the self removal lesion portion of sufferer,
Fine particle is prepared, function of surface is carried out to collagem membrane in collagen film surface and is modified, high-intensitive collagem membrane can be improved
Physics, biological stability, have higher water imbibition, rehydration and water-retaining property, improve the physiological function of cornea, make modified
Collagem membrane implantation sufferer glasses after have it is good the transparency and moisture content, can clinically restore faster the transparency,
Have good bio-compatible performance and provides good bracket for the keratocyte culture of corneal transplantation receptor.In collagen film preparation
In the process, by saltouing, drying and be crosslinked, collagen film-strength obtained is higher than traditional collagen film, after water suction
Can free bend and folding so that corneal restoration plant piece have good mechanics and mechanical performance.Thereby, it is possible to be cornea receptor
Keratocyte growth substrate is provided and realizes cornea reconstruction, solves the technical issues of corneal donor shortage, corneal blindness patient can
Piece is planted based on the corneal restoration that this case provides to cast off illiteracy by corneal graft.
Biologically active corneal restoration based on proposition plants the preparation method of piece, and the present invention also proposes a kind of to use institute
The biologically active corneal restoration for stating preparation method preparation plants piece.
It is described in detail below with a specific embodiment and prepares biologically active angle using above-mentioned preparation method
The process of film reparation plant piece:
1, it uses fresh beef tendon for raw material, is sufficiently cleaned in deionized water, and remove surrounding tissue and fascia, cruelly
Reveal beef tendon, is further sufficiently freezed in refrigerator after washing.
Beef tendon slicer slices across is removed, about 1 millimeter of thickness, suitable vessel is moved into after weighing, is added by weight dense
Degree carries out enzymatic treatment for 0.1% ficin solution, is sufficiently stirred when being set in the insulating box for be placed on 37 ± 1 °
Between proteolysis reaction, timing agitation during reaction.
It is added excessive hydrogen peroxide enzymolysis reaction after setting time, and by the beef tendon of enzymolysis reaction
Tissue is using being dried to obtain collagen after distilled water flushing 3-4 times.
Obtained collagen is swollen using the malonic acid solution of 0.3%-0.5%, mixing is centrifuged off impurity, obtains
The collagen swelling solution of 5g/L.
2, the collagen swelling solution that concentration is 5g/L is mixed with saturated potassium chloride solution, is stirred continuously, so that collagen is gradually
It is precipitated out from collagen swelling solution.And be centrifugated the solution with 12000 revs/min of revolving speed, supernatant is cleaned, it is heavy to collect
The collagen in shallow lake, then extrusion dehydration finally obtain the collagen gel that solid content is about 50mg/L.
Collagen gel is poured into mold, is freeze-dried after cryogenic freezing.
The collagen gel of freeze-drying is placed between two polytetrafluoroethylenes to be flattened with smooth objects such as Kun and is allowed to
As dense collagenous layer.
Dense collagenous layer is put into after being crosslinked 12 hours in 5%EDC solution, rinsing removal crosslinking agent dries to obtain collagen
Film.
Obtained collagen film-strength is higher than traditional collagen film, after absorption can free bend and folding, have good
Mechanics and mechanical performance.
3, by the corneal stroma containing competent cell of the removal lesion portion of autologous patient aseptically in culture dish
Middle chopping, is transferred in the vial disinfected, and clostridiopetidase A is added, sufficiently shreds, and is prepared into micro-granulating device
Even fine particle, particle diameter are centrifuged at 100-300 microns, collect, and biological fibrin glue is added, aseptically will be micro-
Organized corneal material is attached to collagen film surface using 3D printing technique, realizes to the function modified of collagem membrane；It can improve
Physics, the biological stability of high-intensitive collagem membrane have higher water imbibition, rehydration and water-retaining property, improve the physiology of cornea
Function has the good transparency and moisture content after making modified collagem membrane implantation sufferer glasses, clinically can be faster
Recovery the transparency, have good bio-compatible performance, for corneal transplantation receptor keratocyte culture provide it is good
Bracket.
The processing step that the preparation method that aforementioned present invention proposes uses does not influence the molecular structure and characteristic of collagen itself,
The distinctive architectural characteristic of collagen is maintained, the mechanical property of collagem membrane is improved, it can free bend and folding after water suction.Using certainly
The fine particle of body corneal stroma preparation is modified to make collagem membrane have good physics, biological stability, and has higher
Water imbibition, rehydration and water-retaining property, there is the transparency and moisture content well after implantation, can the fast quick-recovery transparency, be cornea
Receptor provides keratocyte growth substrate to realize cornea reconstruction.
It should be noted that the above description is not a limitation of the present invention, the present invention is also not limited to the example above,
The variations, modifications, additions or substitutions that those skilled in the art are made within the essential scope of the present invention, are also answered
It belongs to the scope of protection of the present invention.
Claims (9)
1. the preparation method that biologically active corneal restoration plants piece, which comprises the steps of:
Prepare collagen swelling solution；
Based on the collagen swelling solution, collagem membrane is prepared；
Aseptically, increase surface-functionalized modified material in the collagen film surface in a manner of 3D printing or coating,
Piece is planted to form biologically active corneal restoration；
The preparation step of the surface-functionalized modified material includes:
The corneal stroma containing competent cell of the self removal lesion portion of sufferer is aseptically shredded in culture dish,
It is transferred in the glassware disinfected, increases clostridiopetidase A；
After the corneal stroma for increasing the chopping of clostridiopetidase A is sufficiently shredded, it is prepared into micro-granulating device uniform thin
Microparticle；
After fine particle centrifugation, collecting, biological fibrin glue is added, forms the surface-functionalized modified material.
2. the preparation method that biologically active corneal restoration according to claim 1 plants piece, which is characterized in that described
Collagen swelling solution is prepared, specifically:
Collagen is extracted with protease digestion method in animal knot hoof tissue；
The collagen of extraction is swollen using the malonic acid solution of 0.3%-0.5%, and mixes and is centrifuged off impurity, obtains collagen
Swelling solution.
3. the preparation method that biologically active corneal restoration according to claim 2 plants piece, which is characterized in that described
Collagen is extracted with protease digestion method in animal knot hoof tissue, specifically:
Protein enzyme solution is added by weight in the animal knot hoof tissue；
Stirring is placed on the proteolysis reaction that setting time is carried out in isoperibol, during which timing agitation；
The enzymolysis reaction after setting time；
The animal knot hoof tissue of enzymolysis reaction using distilled water flushing and is dried to obtain collagen.
4. the preparation method that biologically active corneal restoration according to claim 1 plants piece, which is characterized in that described
Collagem membrane is prepared, specifically:
The collagen swelling solution is saltoutd concentration by being saturated inorganic salt solution, obtains collagen aaerosol solution；
The collagen aaerosol solution is subjected to centrifugation and precipitation and separation, obtains collagen gel；
After the collagen gel is placed in mold, cryogenic freezing is freeze-dried；
The collagen gel of freeze-drying is placed between two polytetrafluoroethylenes to be flattened with smooth object and becomes fine and close glue
Former layer；
The dense collagenous layer flattened is subjected to crosslinking Treatment and obtains collagem membrane.
5. the preparation method that biologically active corneal restoration according to claim 2 plants piece, which is characterized in that described
Protease is the ficin that concentration is 0.01%-0.5%.
6. the preparation method that biologically active corneal restoration according to claim 4 plants piece, which is characterized in that described
Crosslinker solution used in crosslinking Treatment is that glutaraldehyde solution, EDC solution, glutamine transaminage solution or epoxy alkyl are molten
Liquid；The mass concentration of the crosslinker solution is 1.5%-25%.
7. the preparation method that biologically active corneal restoration according to claim 1 plants piece, which is characterized in that described
Fine particle diameter is 100-300 microns.
8. the preparation method that biologically active corneal restoration according to claim 1 plants piece, which is characterized in that described
The main component of biological fibrin glue is fibrin, fibrin ferment and ionic calcium soln.
9. a kind of biologically active corneal restoration plants piece, which is characterized in that described in any item according to such as claim 1-8
The preparation method that biologically active corneal restoration plants piece is prepared.
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| CN106390195B (en) * | 2016-11-16 | 2019-08-23 | 四川大学 | A kind of method of modifying of collagem membrane |
| CN109069696B (en) * | 2016-12-16 | 2022-02-01 | 厦门大开生物科技有限公司 | Acellular pig lamellar dry cornea and use method and application thereof |
| CN112494710B (en) * | 2019-09-16 | 2021-12-14 | 天津大学 | Transglutamineenzymatic crosslinked double-network adhesive and preparation method thereof |
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| CN1460715A (en) * | 2003-05-22 | 2003-12-10 | 上海第二医科大学附属第九人民医院 | Production method of transplanting material |
| CN103272268A (en) * | 2013-05-16 | 2013-09-04 | 华南理工大学 | Antibacterial cornea repairing material and preparation method thereof |
| CN103550826A (en) * | 2013-10-31 | 2014-02-05 | 广州优得清生物科技有限公司 | Method for preparing heterogenic cornea material |
| CN105688282A (en) * | 2016-03-11 | 2016-06-22 | 广州宏畅生物科技有限公司 | Novel biological artificial cornea capable of realizing cellularization through in-vivo induction as well as realizing quick transparency |
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| KR101319227B1 (en) * | 2003-10-10 | 2013-10-16 | 게 밍 루이 | Methods and Compositions for Growing Corneal Endothelial and Related Cells on Biopolymers and Creation of Artifical Corneal Transplants |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1460715A (en) * | 2003-05-22 | 2003-12-10 | 上海第二医科大学附属第九人民医院 | Production method of transplanting material |
| CN103272268A (en) * | 2013-05-16 | 2013-09-04 | 华南理工大学 | Antibacterial cornea repairing material and preparation method thereof |
| CN103550826A (en) * | 2013-10-31 | 2014-02-05 | 广州优得清生物科技有限公司 | Method for preparing heterogenic cornea material |
| CN105688282A (en) * | 2016-03-11 | 2016-06-22 | 广州宏畅生物科技有限公司 | Novel biological artificial cornea capable of realizing cellularization through in-vivo induction as well as realizing quick transparency |
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