CN109568663A - The preparation method and cornea scaffold of cornea scaffold - Google Patents
The preparation method and cornea scaffold of cornea scaffold Download PDFInfo
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- CN109568663A CN109568663A CN201811429867.3A CN201811429867A CN109568663A CN 109568663 A CN109568663 A CN 109568663A CN 201811429867 A CN201811429867 A CN 201811429867A CN 109568663 A CN109568663 A CN 109568663A
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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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- 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
- A61L27/3683—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
- A61L27/3687—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 characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
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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
- A61L27/3683—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
- A61L27/3691—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 characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
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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
Abstract
The present invention provides a kind of preparation method of cornea scaffold and cornea scaffolds; it is related to tissue engineering technique field; the preparation method of cornea scaffold provided by the invention; by people's corneal stroma homogenate solution after multigelation through de- cell post-processing molding; this method simple process; medical resource is rationally utilized, large-scale production and clinical application are suitable for.This method is raw material using corneal stroma of the same race homogenate, reconstructs biological response of synthetic materials used for keratoprosthesis on the basis of retaining cornea natural extracellular matrix constituent, and the cornea scaffold being prepared is made to have favorable optical performance, three-dimensional structure and biocompatibility.And remaining antigenic component in cornea is removed by de- cell, rejection occurrence probability after corneal transplantation can be reduced, improves transplanting success rate.
Description
Technical field
The present invention relates to tissue engineering technique fields, more particularly, to the preparation method and cornea of a kind of cornea scaffold
Timbering material.
Background technique
Cornea is the layer of transparent film positioned at eyeball front end, and covering iris, pupil and anterior chamber, well provide greatly for eyes
Part refractive power.Since cornea is directly contacted with external environment, therefore it is highly prone to mechanical trauma, thermal burn and the sense of microorganism
Dye etc. causes cornea that lesion occurs, and less serious case causes corneal transparence decline to influence patient's vision, and serious person will cause the opacity of the cornea
And make blindness.It is counted according to WHO, there is nearly 5,000,000 people in nearly 60,000,000 people of the blind patient of whole world keratonosus, China at present, big absolutely
Most of patients can be cured by corneal transplantation, but cause Most patients cannot get since the height plaque for contributing cornea is weary
Corneal transplantation and can not recover lost eyesight.In recent years, cornea histoengineering rise with development be tissue engineering comea reconstruction in vitro and
Its clinical application creates condition, and also being seen light again for the blind patient of keratonosus by the transplanting of tissue engineering comea, it is new to bring
Wish, but how to obtain the hot and difficult issue that ideal carrier bracket is still the research of current organization engineering cornea reconstruction in vitro.
In terms of carrier bracket, although domestic and foreign scholars to natural biologic material (including contribute people's corneal stroma, collagen,
Gelatin, amnion, fibroin albumen, chitosan, fibrin, entocornea and xenogenesis cell-eliminating coanea matrix etc.), artificial close
At material (including gather light guanidine-acetic acid, polylactic acid one gathers light acetic acid copolymer etc.) and composite material (i.e. natural material and people
Geosynthetics are crosslinked and are integrated by certain ratio or specific method) etc. conduct extensive research and visit
Rope, but can really be made meet tissue engineering comea scale reconstruction in vitro needs carrier bracket it is few, and exist certain
The shortcomings that and deficiency.Utilize tissue engineering comea carrier bracket made of big so large biological molecule such as collagen, gelatin, fibroin albumen
There are still have the defects of structural compactness is insufficient, mechanical property is not good enough, degradation is too fast；Organizational project made of synthetic material
There are the biocompatibility larger disadvantages of ideal, immunogenicity not enough for cornea carrier bracket.In recent years, closest to natural shape
The natural biologic materials such as xenogenesis (animal origins such as pig) corneal stroma of state handle manufactured tissue engineering comea through de- cell and carry
Body support frame becomes research hotspot, but its potential rejection, different plant species corneal curvature difference and ethics problem, makes it forever
It is difficult to become the optimum carrier material received by compatriots.
It can be seen that how to prepare the cornea scaffold with good transparency, three-dimensional structure and biocompatibility still
The emphasis of right tissue engineering comea scale reconstruction in vitro.
In view of this, the present invention is specifically proposed.
Summary of the invention
The first purpose of this invention is to provide a kind of preparation method of cornea scaffold, this method simple process,
Medical resource is rationally utilized, is suitable for large-scale production and clinical application, and the cornea scaffold being prepared is with good
Good optical property, three-dimensional structure and biocompatibility.
Second object of the present invention is to provide the cornea scaffold being prepared using above-mentioned preparation method, the angle
Membrane support material retains someone's cornea natural extracellular matrix constituent, has favorable optical performance, three-dimensional structure and biology
Compatibility.
The present invention provides a kind of preparation method of cornea scaffold, the preparation method includes:
By the homogenate solution of people's corneal stroma through de- cell post-processing molding, the cornea scaffold is obtained；
Wherein, people's corneal stroma is moved from donor's cornea residue tissue, lamellar cornea after penetrating keratoplasty
Plant postoperative donor's cornea residue tissue or full femtosecond laser post-operative cornea matrix lens.
Further, the donor's cornea remaining set is woven to pretreated donor's cornea residue tissue；
Preferably, the pretreatment includes the sclera and conjunctiva that donor's cornea residue tissue is removed to cornea periphery, and clear
It washes；
Preferably, it is cleaned using sterile PBS solution.
Further, the multigelation is that people's corneal stroma is placed in 45~75s of cooling at -180~-200 DEG C
Afterwards, then natural rewarming is to 20-25 DEG C；
After completing the rewarming every time, then carry out cooling next time；
Preferably, the number of the cooling and the rewarming is no less than 3 times.
Further, people's corneal stroma after the multigelation is ground to powdered at -180~-200 DEG C, added
The homogenate solution is made in water；
Wherein, people's corneal stroma is ground to 150-250 mesh, preferably 170-230 mesh；
Preferably, people's corneal stroma is ground using beveller；
Preferably, after grinding 20-40s to people's corneal stroma using beveller, 20-40s is pre-chilled；
After completing the pre-cooling every time, then carry out grinding next time.
Further, DNase and RNase is added in Xiang Suoshu homogenate solution and carries out de- cell；
Preferably, 40-60U/ml DNase and 0.5-2U/ml RNase is added and carries out de- cell；
Preferably, cleaning removal of impurities then reprocessing molding is carried out after further including the steps that de- cell；
Preferably, it is cleaned with ultrapure water, is cleaned in eccentric fashion；
Preferably, it is cleaned with 8000-12000rpm centrifugation 20-40min, it is final to collect precipitating.
Further, the processing, which forms, includes:
Homogenate solution after de- cell is lyophilized under vacuum environment, obtains white sponge substance, by the white sea
Continuous shape substance is dissolved in the cornea tissue suspension that water obtains and is added in mold, air-dries and then forms；
Preferably, the concentration of the cornea tissue suspension be 1-100mg/ml, preferably 2-80mg/ml, more preferably
5-60mg/ml；
Preferably, it is air-dried in constant-temperature constant-humidity environment；
Preferably, the temperature of the constant temperature is 20-30 DEG C, preferably 22-28 DEG C, more preferably 25-27 DEG C；
Preferably, the humidity of the constant humidity is 70%-95%, preferably 75%-90%, more preferably 80%-85%；
Preferably, the air-dried time is 60-80 hour.
Further, further include the steps that cross-linking reaction after processing molding；
Preferably, cornea scaffold after molding is soaked in 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide
Cross-linking reaction is carried out in n-hydroxysuccinimide cross-linking reaction aqueous solution；
Preferably, the temperature of the cross-linking reaction is 3-5 DEG C；
Preferably, the time of the cross-linking reaction is 20-30 hour.
Further, cleaning is further included the steps that after cross-linking reaction；
Preferably, the cornea scaffold is cleaned 10-15 hours with ultrapure water；
Preferably, every 20-40min replaces a ultrapure water in the process of cleaning.
Further, further include the steps that the cornea scaffold that will be obtained sterilizes；
Preferably, it is sterilized using physical method, it is preferred to use the method for radiation exposure sterilizes, and more preferably uses
Co 60 irradiation sterilizes.
In addition, being prepared the present invention also provides a kind of cornea scaffold using above-mentioned preparation method.
The preparation method of cornea scaffold provided by the invention, by the homogenate solution of people's corneal stroma after de- cell
Reason molding, obtains cornea scaffold.This method simple process rationally utilizes medical resource, is suitable for large-scale production and clinic
It promotes and applies.The homogenate of this method user's corneal stroma is raw material, in the base for retaining cornea natural extracellular matrix constituent
Biological response of synthetic materials used for keratoprosthesis is reconstructed on plinth, on the one hand makes the cornea scaffold being prepared that there is favorable optical performance, three
Tie up structure and biocompatibility.On the other hand, remaining antigenic component in cornea is removed by de- cell, corneal transplantation can be reduced
Postoperative rejection occurrence probability improves transplanting success rate.
In addition, the present invention also provides the cornea scaffold being prepared using above-mentioned preparation method, the corneal stent
Material remains with cornea natural extracellular matrix constituent, has favorable optical performance, three-dimensional structure and biocompatibility.Together
When, additionally it is possible to rejection occurrence probability after corneal transplantation is reduced, transplanting success rate is improved.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is the transparency results figure for the cornea scaffold that experimental example 2 of the present invention provides；
Fig. 2 is the HE coloration result figure for the cornea scaffold that experimental example 2 of the present invention provides.
Specific embodiment
Technical solution of the present invention is clearly and completely described below in conjunction with attached drawing, it is clear that described implementation
Example is a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, ordinary skill
Personnel's every other embodiment obtained without making creative work, shall fall within the protection scope of the present invention.
The present invention provides a kind of preparation methods of cornea scaffold, comprising:
By the homogenate solution of people's corneal stroma through de- cell post-processing molding, cornea scaffold is obtained；
Wherein, people's corneal stroma is donor's cornea residue tissue, lamellar keratoplasty after penetrating keratoplasty
Donor's cornea residue tissue or full femtosecond laser post-operative cornea matrix lens afterwards.
The preparation method of cornea scaffold provided by the invention, this method simple process rationally utilize medical resource, fit
In large-scale production and clinical application.This method is raw material using hetero stroma of cornea homogenate, is retaining cornea natural fine
Biological response of synthetic materials used for keratoprosthesis is reconstructed on the basis of extracellular matrix constituent, on the one hand makes the cornea scaffold being prepared
With favorable optical performance, three-dimensional structure and biocompatibility.On the other hand, remaining antigen in cornea is removed by de- cell
Ingredient can reduce rejection occurrence probability after corneal transplantation, improve transplanting success rate.
In some preferred embodiments, donor's cornea remaining set is woven to pretreated donor's cornea residue tissue.
Preferably, the pretreatment includes the sclera and conjunctiva that donor's cornea residue tissue is removed to cornea periphery, and clear
It washes.
The sclera and conjunctiva for removing cornea periphery, can make the cornea scaffold being prepared only retain cornea natural fine
Extracellular matrix constituent, and other constituent impurities are free of, guarantee the properties of cornea scaffold.
Preferably, it is cleaned using sterile PBS solution, is preferably cleaned three times.
In some preferred embodiments, multigelation is that people's corneal stroma is placed in -180~-200 DEG C of declines
After 45~75s of temperature, then natural rewarming is to 20-25 DEG C.
Wherein, the environment temperature of cooling for example can be, but be not limited to -180 DEG C, -185 DEG C, -190 DEG C, -195 DEG C or -
200℃.The time of cooling for example can be, but be not limited to 45s, 50s, 55s, 60s, 65s or 70s.The environment temperature example of rewarming
It such as can be, but be not limited to 20 DEG C, 21 DEG C, 22 DEG C, 23 DEG C, 24 DEG C or 25 DEG C.
After completing rewarming every time, then carry out cooling next time.
Wherein, cool down identical as the number of rewarming.
Preferably, cool down and the number of rewarming is no less than 3 times, such as can be, but be not limited to 3 times, 4 times, 5 times or 6 times.
Multigelation can make the water in cell form crystallization, cause salinity in remaining cell liquid to increase, due to cell
Interior ice pellets is formed, and increasing for salinity can make cell rupture in remaining cell liquid, to reach only reservation cornea n cell
The purpose of epimatrix constituent.
It is preferable to use PBS solution rinsings, carry out rewarming to people's corneal stroma.
In some preferred embodiments, people's corneal stroma after multigelation is ground at -180~-200 DEG C
It is powdered, add water that the homogenate solution is made.
Wherein, the environment temperature of grinding for example can be, but be not limited to -180 DEG C, -185 DEG C, -190 DEG C, -195 DEG C or -
200℃。
Wherein, people's corneal stroma is ground to 150-250 mesh, such as can be, but be not limited to 150 mesh, 160 mesh,
170 mesh, 180 mesh, 190 mesh, 200 mesh, 210 mesh, 220 mesh, 230 mesh, 240 mesh or 250 mesh, preferably 170-230 mesh.
Preferably, people from institute's corneal stroma is ground using beveller.
It is ground using beveller, grinding can be made more sufficiently, more thoroughly, and effectively save human cost and time
Cost.Commercially available beveller.
Preferably, after grinding 20-40s to people's corneal stroma using beveller, 20-40s is pre-chilled.
Wherein, milling time for example can be, but be not limited to 20s, 25s, 30s, 35s or 40s, and pre-coo time for example can be with
For, but it is not limited to 20s, 25s, 30s, 35s or 40s.
After completing the pre-cooling every time, then carry out grinding next time.
Grinding is combined with pre-cooling, can guarantee that environment temperature when grinding is maintained at -180~-200 DEG C of low temperature ring
Under border, avoid because of the heat production that rubs.
In some preferred embodiments, DNase and RNase is added into homogenate solution and carries out de- cell.
DNase, that is, deoxyribonuclease, Chinese are deoxyribonuclease, and it is single-stranded to be that one kind can digest
Or double-stranded DNA generates the endonuclease of monodeoxyribonucleotide or single-stranded or double-stranded oligodeoxynucleotide.RNase is
Ribonuclease, Chinese are ribalgilase, are the nucleases that a kind of catalysis RNA is degraded to small fragment.
De- cell is carried out using above two enzyme to handle, and the antigenicity for the cornea scaffold being prepared can be made small,
Avoid rejection.
Preferably, 40-60U/ml DNase and 0.5-2U/ml RNase is added and carries out de- cell.
Wherein, the additional amount of DNase for example can be, but be not limited to 40U/ml, 45U/ml, 50U/ml, 55U/ml or
60U/ml；The additional amount of RNase for example can be, but be not limited to 0.5U/ml, 1U/ml, 1.5U/ml or 2U/ml.
Preferably, cleaning removal of impurities then reprocessing molding is carried out after further including the steps that de- cell.
It is cleaned and is cleaned, can guarantee preferably to take off cell effect.
Preferably, it is cleaned with ultrapure water, is cleaned in eccentric fashion；
Preferably, it is cleaned with 8000-12000rpm centrifugation 20-40min, it is final to collect precipitating.
Wherein, the speed of centrifugation for example can be, but be not limited to 8000rpm, 9000rpm, 10000rpm, 11000rpm or
12000rpm, the time of centrifugation for example can be, but be not limited to 20min, 25min, 30min, 35min or 40min.
In some preferred embodiments, processing, which forms, includes:
Homogenate solution after de- cell is lyophilized under vacuum environment, obtains white sponge substance, by the white sea
Continuous shape substance is dissolved in the cornea tissue suspension that water obtains and is added in mold, air-dries and then forms.
Preferably, the concentration of the cornea tissue suspension is 1-100mg/ml, such as can be, but be not limited to 1mg/
Ml, 10mg/ml, 20mg/ml, 30mg/ml, 40mg/ml, 50mg/ml, 60mg/ml, 70mg/ml, 80mg/ml, 90mg/ml or
100mg/ml, preferably 2-80mg/ml, more preferably 5-60mg/ml；
Molding can position according to cornea lesion and size personalized customization cornea scaffold, pass through and change cornea tissue
Suspension concentration produces the timbering material of different-thickness demand respectively, for such as infected keratitis, keratoconus, cornea
The diseases such as endothelium decompensation, which drill through, plants piece size needed for lesion range.
Preferably, it is air-dried in constant-temperature constant-humidity environment.
Preferably, the temperature of the constant temperature is 20-30 DEG C, such as can be, but be not limited to 20 DEG C, 21 DEG C, 22 DEG C, 23
DEG C, 24 DEG C, 25 DEG C, 26 DEG C, 27 DEG C, 28 DEG C, 29 DEG C or 30 DEG C, preferably 22-28 DEG C, more preferably 25-27 DEG C；
Preferably, the humidity of the constant humidity is 70%-95%, such as can be, but be not limited to 70%, 75%, 80%,
85%, 90% or 95%, preferably 75%-90%, more preferably 80%-85%；
Preferably, the air-dried time is 60-80 hour, such as can be, but is not limited to 60 hours, 65 small
When, 70 hours, 75 hours or 80 hours.
In some preferred embodiments, further include the steps that cross-linking reaction after processing molding.
Preferably, cornea scaffold after molding is soaked in 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide
Cross-linking reaction is carried out in n-hydroxysuccinimide cross-linking reaction aqueous solution.
EDC (1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride) is a water-soluble carbodiimide, is used
In protein and nucleic acid crosslinking and producing for coupled object is immunized, be used in conjunction, can be improved with NHS (n-hydroxysuccinimide)
Coupling efficiency.
Use the aqueous solution of 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide and n-hydroxysuccinimide as friendship
Join reaction solution, can effectively promote the mechanical property of cornea scaffold.
Preferably, the temperature of the cross-linking reaction is 3-5 DEG C, such as can be, but be not limited to 3 DEG C, 4 DEG C or 5 DEG C.
Preferably, the time of the cross-linking reaction is 20-30 hour, such as can be, but be not limited to 20 hours, 22
A hour, 24 hours, 26 hours, 28 hours or 30 hours.
In some preferred embodiments, cleaning is further included the steps that after cross-linking reaction；
Preferably, the cornea scaffold is cleaned 10-15 hours with ultrapure water.
Wherein, ultrapure water cleaning time for example can be, but be not limited to 10 hours, 11 hours, 12 hours, 13 hours,
14 hours or 15 hours.
Preferably, every 20-40min replaces a cleaning solution in the process of cleaning, such as can be, but be not limited to 20min,
25min, 30min, 35min or 40min.
In some preferred embodiments, further include the steps that the cornea scaffold that will be obtained sterilizes.
Sterilizing can guarantee that cornea scaffold is germ-free condition, convenient for storage and use.
Preferably, it is sterilized using physical method, it is preferred to use the method for radiation exposure sterilizes, and more preferably uses
Co 60 irradiation sterilizes.
It is sterilized, is avoided using any toxic chemical reagent or drug, safety is higher using physical method.
In addition, being prepared the present invention also provides a kind of cornea scaffold using above-mentioned preparation method.
The cornea scaffold remains with cornea natural extracellular matrix constituent, has favorable optical performance, three-dimensional
Structure and biocompatibility.Simultaneously, additionally it is possible to reduce rejection occurrence probability after corneal transplantation, improve transplanting success rate.
The present invention is further detailed below with reference to embodiment.
Embodiment 1
A kind of cornea scaffold is present embodiments provided, preparation method includes the following steps:
(a), under the disecting microscope in Biohazard Safety Equipment, donor's cornea residue tissue after penetrating keratoplasty is gone
It except the sclera and conjunctiva on cornea periphery, is washed three times with sterile PBS solution (1 ×), then the cornea tissue after preliminary treatment is placed in
Ultra low temperature freezer storage.
(b), it takes out with sterile PBS solution (1 ×) rinsing rewarming, then is placed in -180 DEG C of low temperature environments, rewarming again after 75s
To 20 DEG C, it is repeated 3 times, it is then clean with sterile PBS repeated flushing.
(c), -180 DEG C of low temperature environments of merging in grinding container are put into after removing surface moisture, are transported with beveller 20s
Row --- 20s is pre-chilled, and is broken into powder after three circulations, and ultrapure water is added and is mixed into homogenate solution, the concentration of homogenate solution is
1mg/ml。
(d), 40U/ml DNase I and 2U/ml RNase 2h is added and carries out de- cell processing, then use ultrapure water with
8000rpm 40min is cleaned three times repeatedly, collects final precipitating.
(e), it is placed in 20h in -80 DEG C and is put into vacuum freeze drier after freezing reality, be lyophilized into white sponge.
(f), the spongy material after freeze-drying is dissolved in ultrapure water, magnetic agitation 4h obtains cornea tissue suspension.
(g), cornea tissue suspension is added in mold, 20 DEG C of Yu Wendu, air-dries 3 days, obtains in 95% environment of humidity
Cornea scaffold.
(h), EDC/NHS cross-linking reaction aqueous solution is prepared, cornea scaffold is soaked in wherein, is waited in 4 DEG C of placement 20h
Cross-linking reaction is complete.
(i), cornea scaffold is cleaned into 1h with ultrapure water, every 40min replaces a cleaning solution.
(j), Co 60 sterilizes after sealing, and saves backup at -20 DEG C.
Embodiment 2
A kind of cornea scaffold is present embodiments provided, preparation method includes the following steps:
(a), under the disecting microscope in Biohazard Safety Equipment, donor's cornea residue tissue after lamellar keratoplasty is gone
It except the sclera and conjunctiva on cornea periphery, is washed three times with sterile PBS solution (1 ×), then the cornea tissue after preliminary treatment is placed in
Ultra low temperature freezer storage.
(b), it takes out with sterile PBS solution (1 ×) rinsing rewarming, then is placed in -200 DEG C of low temperature environments, rewarming again after 45s
To 25 DEG C, it is repeated 3 times, it is then clean with sterile PBS repeated flushing.
(c), -200 DEG C of low temperature environments of merging in grinding container are put into after removing surface moisture, are transported with beveller 40s
Row --- 40s is pre-chilled, and is broken into powder after three circulations, and ultrapure water is added and is mixed into homogenate solution, the concentration of homogenate solution is
100mg/ml。
(d), 60U/ml DNase I and 0.5U/ml RNase 2h is added and carries out de- cell processing, then use ultrapure water with
12000rpm 20min is cleaned three times repeatedly, collects final precipitating.
(e), it is placed in 30h in -80 DEG C and is put into vacuum freeze drier after freezing reality, be lyophilized into white sponge.
(f), the spongy material after freeze-drying is dissolved in ultrapure water, magnetic agitation 4h obtains cornea tissue suspension.
(g), cornea tissue suspension is added in mold, 30 DEG C of Yu Wendu, air-dries 3 days, obtains in 70% environment of humidity
Cornea scaffold.
(h), EDC/NHS cross-linking reaction aqueous solution is prepared, cornea scaffold is soaked in wherein, is waited in 4 DEG C of placement 30h
Cross-linking reaction is complete.
(i), cornea scaffold is cleaned into 3h with ultrapure water, every 20min replaces a cleaning solution.
(j), Co 60 sterilizes after sealing, and saves backup at -20 DEG C.
Embodiment 3
A kind of cornea scaffold is present embodiments provided, preparation method includes the following steps:
(a), under the disecting microscope in Biohazard Safety Equipment, by full femtosecond laser post-operative cornea matrix lens with sterile
PBS solution (1 ×) is washed three times, merging ultra low temperature freezer storage.
(b), take out with sterile PBS solution (1 ×) rinsing rewarming, then be placed in liquid nitrogen, after 1min again rewarming to 22 DEG C,
It is repeated 3 times, it is then clean with sterile PBS repeated flushing.
(c), it is put into grinding container in merging liquid nitrogen after removing surface moisture, is run with beveller 30s --- 30s is pre-
It is cold, it is broken into powder after three circulations, ultrapure water is added and is mixed into homogenate solution, the concentration of homogenate solution is 50mg/ml.
(d), 50U/ml DNase I and 1U/ml RNase 2h is added and carries out de- cell processing, then use ultrapure water with
10000rpm 30min is cleaned three times repeatedly, collects final precipitating.
(e), it is placed in -80 DEG C after freezing reality for 24 hours and is put into vacuum freeze drier, be lyophilized into white sponge.
(f), the spongy material after freeze-drying is dissolved in ultrapure water, magnetic agitation 4h obtains cornea tissue suspension.
(g), cornea tissue suspension is added in mold, 25 DEG C of Yu Wendu, air-dries 3 days, obtains in 80% environment of humidity
Cornea scaffold.
(h), prepare EDC/NHS cross-linking reaction aqueous solution, by cornea scaffold be soaked in wherein, in 4 DEG C place for 24 hours to
Cross-linking reaction is complete.
(i), cornea scaffold is cleaned into 2h with ultrapure water, every 30min replaces a cleaning solution.
(j), Co 60 sterilizes after sealing, and saves backup at -20 DEG C.
Embodiment 4
A kind of cornea scaffold is present embodiments provided, preparation method includes the following steps:
(a), under the disecting microscope in Biohazard Safety Equipment, by full femtosecond laser post-operative cornea matrix lens, use is sterile
PBS solution (1 ×) is washed three times, merging ultra low temperature freezer storage.
(b), it takes out with sterile PBS solution (1 ×) rinsing rewarming, then is placed in -150 DEG C of low temperature environments, rewarming again after 30s
To 30 DEG C, it is repeated 3 times, it is then clean with sterile PBS repeated flushing.
(c), -150 DEG C of low temperature environments of merging in grinding container are put into after removing surface moisture, are transported with beveller 45s
Row --- 45s is pre-chilled, and is broken into powder after three circulations, and ultrapure water is added and is mixed into homogenate solution, the concentration of homogenate solution is
2mg/ml。
(d), 30U/ml DNase I and 3U/ml RNase 2h is added and carries out de- cell processing, then use ultrapure water with
6000rpm 45min is cleaned three times repeatedly, collects final precipitating.
(e), it is placed in -80 DEG C after freezing reality for 24 hours and is put into vacuum freeze drier, be lyophilized into white sponge.
(f), the spongy material after freeze-drying is dissolved in ultrapure water, magnetic agitation 4h obtains cornea tissue suspension.
(g), cornea tissue suspension is added in mold, 15 DEG C of Yu Wendu, air-dries 3 days, obtains in 60% environment of humidity
Cornea scaffold.
(h), EDC/NHS cross-linking reaction aqueous solution is prepared, cornea scaffold is soaked in wherein, is waited in 4 DEG C of placement 15h
Cross-linking reaction is complete.
(i), cornea scaffold is cleaned into 1h with ultrapure water, every 30min replaces a cleaning solution.
(j), Co 60 sterilizes after sealing, and saves backup at -20 DEG C.
Experimental example 1
The cornea scaffold that this experimental example selects embodiment 1 to provide carries out subsequent experimental.
1, transparency is tested
The cornea scaffold that embodiment 3 provides is soaked in saturation water suction in 24 hours in PBS solution, is absorbed with filter paper
Surface moisture is observed its transparent situation, and is photographed to record.
As a result as shown in Figure 1, from figure 1 it appears that the transparency of cornea scaffold provided by the invention is good.
2, HE Coloration experiment
(1) paraffin section: being soaked in saturation water suction in 24 hours in PBS solution for the cornea scaffold of reconstruction, solid through PFA
After fixed, routine paraffin wax embedding, slice.Slice is conventional to be dewaxed with dimethylbenzene, through ethyl alcohol at different levels to washing: dimethylbenzene (I) 5min；Two
Toluene (II) 5min；100% ethyl alcohol 2min；95% ethyl alcohol 1min；80% ethyl alcohol 1min；75% ethyl alcohol 1min；PBS is washed
2min。
(2) haematoxylin dyeing 5min, PBS wash 5min.
(3) acidic alcohol breaks up 30s.
(4) PBS washes 5min three times.
(5) Yihong liquid 2min is set.
(6) conventional dehydration, transparent, mounting: 95% ethyl alcohol (I) 1min；95% ethyl alcohol (II) 5min；100% ethyl alcohol (I)
5min；100% ethyl alcohol (II) 5min；Dimethylbenzene (I) 5min；Dimethylbenzene (II) 5min；Dimethylbenzene (III) 5min；Resinene
Sealing.
(7) optical microphotograph microscopic observation, film making.
As a result as shown in Fig. 2, from figure 2 it can be seen that cornea scaffold provided by the invention, structural integrity is continuous,
De- cell effect is obvious.The process of undergoing reconstruction remains corneal extracellular matrix ingredient.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: its according to
So be possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features into
Row equivalent replacement；And these are modified or replaceed, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme.
Claims (10)
1. a kind of preparation method of cornea scaffold, which is characterized in that the preparation method includes:
By the homogenate solution of people's corneal stroma through de- cell post-processing molding, the cornea scaffold is obtained；
Wherein, people's corneal stroma is from donor's cornea residue tissue, lamellar keratoplasty after penetrating keratoplasty
Donor's cornea residue tissue or full femtosecond laser post-operative cornea matrix lens afterwards.
2. preparation method according to claim 1, which is characterized in that the donor's cornea remaining set is woven to pretreated
Donor's cornea residue tissue；
Preferably, the pretreatment includes the sclera and conjunctiva that donor's cornea residue tissue is removed to cornea periphery, and is cleaned；
Preferably, it is cleaned using sterile PBS solution.
3. preparation method according to claim 1, which is characterized in that the multigelation is to set people's corneal stroma
At -180~-200 DEG C after 45~75s of cooling, then natural rewarming is to 20-25 DEG C；
After completing the rewarming every time, then carry out cooling next time；
Preferably, the number of the cooling and the rewarming is no less than 3 times.
4. preparation method according to claim 1, which is characterized in that by people's corneal stroma after the multigelation-
It is ground to powdered at 180~-200 DEG C, adds water that the homogenate solution is made；
Wherein, people's corneal stroma is ground to 150-250 mesh, preferably 170-230 mesh；
Preferably, people's corneal stroma is ground using beveller；
Preferably, after grinding 20-40s to people's corneal stroma using beveller, 20-40s is pre-chilled；
After completing the pre-cooling every time, then carry out grinding next time.
5. preparation method according to claim 1, which is characterized in that DNase and RNase is added in Xiang Suoshu homogenate solution
Carry out de- cell；
Preferably, 40-60U/ml DNase and 0.5-2U/ml RNase is added and carries out de- cell；
Preferably, cleaning removal of impurities then reprocessing molding is carried out after further including the steps that de- cell；
Preferably, it is cleaned with ultrapure water, is cleaned in eccentric fashion；
Preferably, it is cleaned with 8000-12000rpm centrifugation 20-40min, it is final to collect precipitating.
6. preparation method according to claim 1, which is characterized in that the processing, which forms, includes:
Homogenate solution after de- cell is lyophilized under vacuum environment, obtains white sponge substance, by the white sponge
Substance is dissolved in the cornea tissue suspension that water obtains and is added in mold, air-dries and then forms；
Preferably, the concentration of the cornea tissue suspension is 1-100mg/ml, preferably 2-80mg/ml, more preferably 5-
60mg/ml；
Preferably, it is air-dried in constant-temperature constant-humidity environment；
Preferably, the temperature of the constant temperature is 20-30 DEG C, preferably 22-28 DEG C, more preferably 25-27 DEG C；
Preferably, the humidity of the constant humidity is 70%-95%, preferably 75%-90%, more preferably 80%-85%；
Preferably, the air-dried time is 60-80 hour.
7. preparation method according to claim 1, which is characterized in that processing molding after further include cross-linking reaction step
Suddenly；
Preferably, cornea scaffold after molding is soaked in 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide and N-
Cross-linking reaction is carried out in hydroxysuccinimide crosslinker reacting solution；
Preferably, the temperature of the cross-linking reaction is 3-5 DEG C；
Preferably, the time of the cross-linking reaction is 20-30 hour.
8. preparation method according to claim 7, which is characterized in that further include the steps that cleaning after cross-linking reaction；
Preferably, the cornea scaffold is cleaned 10-15 hours with ultrapure water；
Preferably, every 20-40min replaces a ultrapure water in the process of cleaning.
9. preparation method according to claim 1-8, which is characterized in that further include the corneal stent material that will be obtained
The step of material is sterilized；
Preferably, it is sterilized using physical method, it is preferred to use the method for radiation exposure sterilizes, and more preferably uses Co 60
Irradiation sterilizes.
10. a kind of cornea scaffold, which is characterized in that be prepared into using the described in any item preparation methods of claim 1-9
It arrives.
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