CN1579342A - Exogenous cornea substrate without cells and its preparation method and use - Google Patents
Exogenous cornea substrate without cells and its preparation method and use Download PDFInfo
- Publication number
- CN1579342A CN1579342A CN 200410018107 CN200410018107A CN1579342A CN 1579342 A CN1579342 A CN 1579342A CN 200410018107 CN200410018107 CN 200410018107 CN 200410018107 A CN200410018107 A CN 200410018107A CN 1579342 A CN1579342 A CN 1579342A
- Authority
- CN
- China
- Prior art keywords
- cornea
- acellular
- stroma
- hetero
- corneal
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Abstract
The invention provides a cell-less heterogenetic cornea material and manufacturing method and use, the manufacturing method for the product is: acquires the fresh animals' cornea at first; eliminates the cell membrane of the cornea material and nucleic acid with chemical method and enzyme method; carries on dehydration process to the cell-less heterogenetic cornea material; stores for backup. The method eliminates the antigenicity of the heterogenetic cornea material through eliminating the cell elements in the cornea material. At the same time, it reserves the integrity of glue fiber and transparency of the cornea. The material can be applied to construction of manmade cornea or used as the medical material for cornea transplantation, and cornea refraction operation directly.
Description
Technical field
The invention belongs to medical material, relate to a kind of corneal stroma, relate in particular to a kind of acellular hetero stroma of cornea and preparation method, the artificial cornea that this acellular hetero stroma of cornea both can be used for organizational project makes up, and can be used as medical material again and is used for corneal transplantation, refractive surgery.
Background technology
Keratopathy is the second largest disease that causes blinding in the global range.
1For the patient of keratopathy blinding, have only by corneal transplantation just to make the patient recover vision.In China,, and accept only thousands of examples in patient's every year of corneal transplantation because of annual nearly 250,000 examples of the patient of keratopathy blinding.The basic reason that the restriction corneal transplantation is carried out is the extreme scarcity in donor's cornea source.Therefore, researching and developing out the cornea substitute that can substitute people's cornea is the key that solves this imbalance between supply and demand.
The history of the existing many decades of artificial cornea's research
2, the research in past mainly concentrates on the materialogy aspect
3Because people's cornea is the part of wall of eyeball, not only requiring concerning graft substitute has strict optical characteristics, also must possess simultaneously and the highly organized biocompatibility of human eye.In recent years, the structure that develops into the artificial cornea of tissue engineering technique provides new thinking and method.Minami etc.
5Is the reconstruction in vitro that substrate has realized the artificial cornea in 1993 with the collagen gel, but fails to be used for transplant operation owing to the transparency difference of gel and intensity are low.Griffith in 1999 etc.
6Genetically modified endothelium and epithelial cell are cultivated on the substrate based on collagen-chondroitin sulfate, rebuild all similar biological cornea of function, structure to normal cornea, but also fail to enter animal experiment stage, just differ farther from clinical practice because matrix strength is not enough.Therefore, the cornea biomaterial of synthetic is also very undesirable so far
4Up to now relevant artificial cornea's biomaterial is studied topmost limitation and is, can't prepare the substrate framework that has the similar transparency, intensity and biological characteristics to people's cornea.
Hetero stroma of cornea such as porcine cornea substrate have and the similar organizational structure of people's corneal stroma, biophysical properties and optical characteristics
7But intensive rejection and xenotransplantation may make infectious agent infect human body after the xenotransplantation, and this two big factor causes the heterogenic cornea transplanting can't use clinically so far
8Stromal cell in the discovering in recent years, corneal stroma is the major antigen that causes the matrix type rejection
9, also be the carrier of infectious agent such as virus.And as collagen fiber high conservative between kind of system of corneal stroma framework, the difference of organizational structure and biophysical properties is little, and antigenicity is very low, and acellular heterogenic cornea does not produce rejection after transplanting.Therefore, the stromal cell of animal corneal is sloughed fully, made it to become the desirable substitute that acellular hetero stroma of cornea may be people's corneal stroma.Rebuild the field at external organ, existing research is passed through the xenogenesis cardiac valve
10, dermal tissue
11, the tendon ligament
12Take off the cell processing in tissue, obtain acellular fiber framework.They have no antigen, advantages such as excellent biological compatibility, and the collagen fiber framework almost can completely keep.Some takes off cell heteroplasm and successfully has been applied to clinical
13
Summary of the invention
An object of the present invention is to provide a kind of acellular hetero stroma of cornea.
Another object of the present invention provides the preparation method of this acellular hetero stroma of cornea, is achieved through the following technical solutions:
(1) obtains the fresh animal corneal stroma: win animal eyeball, prune and to be soaked in after the eyeball surface affiliated group in 5% povidone iodine 3 minutes, wash with 500 ml physiological salines then, used animal mainly comprises pig, cattle, Canis familiaris L., cut the full-shape film along limbus of corneae, tear corneal epithelium and posterior elastic membrane off, obtain animal corneal substrate.Also animal eyeball can be put-70 ℃ of stored frozen earlier, capable again aforesaid operations after the normal temperature unfreezing.
(2) processing of eluting keratocyte film component and removal nucleic acid: 1. the animal corneal substrate that obtains is placed ion-type or nonionic detergent solution, place on the constant temperature shaking table and vibrate, purpose is with keratocyte film component eluting, changed 1 time in the every 6-12 of detergent solution hour, temperature keeps 25 ℃-37 ℃, pH value 6-8, elution time 24-72 hour, 2. the corneal stroma reuse nuclease solution rinsing through 1. handling, purpose is with residual ribonucleic acid and DNA (deoxyribonucleic acid) composition eluting, temperature keeps 27 ℃-37 ℃, pH value 6-8,3. the corneal stroma through 2. handling obtains acellular hetero stroma of cornea with balanced salt solution flushing 48-72 hour.
The solution of detergent is advisable with hypotonic solution, the solution osmotic pressure is that 0mosm is between the 299mosm, ionic detergent preferably sodium dodecyl sulfate (sodium dodecyl sulfate, SDS), the preferred bent ketone X100 of nonionic detergent (Triton X-100), the suitable concentration of sodium lauryl sulphate is between 0.05% to 0.1%, and the suitable concentration of bent ketone X100 is between 0.1% to 4%.In order to reduce the destruction of protease to substrate collagen to greatest extent, (ethylenediaminetetraacetic acid, EDTA), suitable concn is 1-25mmol/L should to add collagenase inhibitors such as diethylamine tetraacethyl in the solution.Nuclease is selected excision enzyme or restriction endonuclease, excision enzyme is selected ribonuclease A (RNaseA) and deoxyribonuclease I (DNaseI), restriction endonuclease is selected EcoR I and Hind III etc., the concentration range of ribonuclease A is that 0.02mg/ml is to 1mg/ml, the concentration range of deoxyribonuclease I is that 0.2mg/ml is to 10mg/ml, for nuclease is played one's part to the full, nuclease solution should be used the preparation of physiological buffer solution, preferred Hank balanced salt solution (Hanks ' balanced salt solution, HBSS).Wash used balanced salt solution preferably phosphoric acid balanced salt solution (Phosphate balanced solution, PBS).
(3) acellular hetero stroma of cornea processed: carry out 12-24 hour processed with pure glycerin.
(4) preserve: the corneal stroma after will dewatering places 85% glycerite to preserve, and is standby.
Another purpose of the present invention is the structure that this acellular hetero stroma of cornea is used for the artificial cornea of organizational project, or direct medical material as corneal transplantation, cornea refractive surgery etc.
Main feature of the present invention is both can remove all cell component in the animal corneal substrate through the above-mentioned cell processing method of taking off, and can preserve the basic framework of corneal collagen fiber again, thereby keep the toughness and the transparency of cornea.Hetero stroma of cornea after the processing has very low immunogenicity and excellent biological compatibility.It can be directly used in, and therapeutic corneal is transplanted and lamellar cornea is transplanted, the corneal epithelium of transplanting the back receptor makes acellular hetero stroma of cornea plant the superficial epitheliumization of sheet very soon, the keratocyte of receptor and the nerve acellular hetero stroma of cornea of can growing into is again planted in the sheet, reaches the purpose of corneal transplantation fully.
Description of drawings
Fig. 1 a sees before freezing porcine cornea takes off the cell processing substantially.
Fig. 1 b sees after porcine cornea takes off the cell processing substantially.
Fig. 2 a is that freezing porcine cornea takes off preceding Lignum Sappan Soviet Union-Yihong (HE) dyeing of cell processing photo.
Fig. 2 b is that porcine cornea takes off cell processing back HE dyeing photo.
Fig. 3 a is that freezing porcine cornea takes off the preceding transmission electron microscope photo of cell processing.
Fig. 3 b is that porcine cornea takes off cell processing back transmission electron microscope photo.
Fig. 4 is with after taking off the lamellar keratoplasty that the cell porcine cornea is a donor.
Fig. 5 transplants at the lamellar cornea of lagophthalmos for being donor to take off the cell porcine cornea, and 4 days epithelization of postoperative are finished.
The specific embodiment
Below will the present invention is further illustrated by embodiment.
Embodiment 1 takes off the preparation of cell porcine cornea substrate
In qualified feed lot, select the healthy donors pig, after penthiobarbital pin intramuscular injection anesthesia, win the Oculus sus domestica ball, prune after the eyeball surface affiliated group and eyeball to be soaked in 5% povidone iodine 3 minutes, wash with 500 ml physiological salines then, be placed on-70 ℃ of stored frozen, handle until taking off cell.
After thawing under the eyeball room temperature, operating microscope lower edge limbus of corneae is cut the full-shape film, scrape off corneal epithelium and tear posterior elastic membrane off, referring to Fig. 1 a, Fig. 2 a, Fig. 3 a, then this corneal stroma is placed the 30ml ionic detergent, place on the constant temperature shaking table with 300 rev/mins of uninterrupted concussions 24 hours, changed solution once in per 6 hours, ionic detergent is selected 0.05%SDS (SIGMA chemical company for use, St.Louis MO) is dissolved in the deionized water, and adds 1mmol/L EDTA, treatment temperature is 37 ℃, and the solution pH value is 8.0.
Take out the hetero stroma of cornea after the above-mentioned processing, with HBSS (Alcon Laboratories, FortWorth, TX) flushing is 1 hour, the residual solution on eccysis surface, place nuclease solution then, with 300 rev/mins of uninterrupted concussions 8 hours, nuclease solution adopted RNaseA 0.02mg/mL, DNaseI 0.2mg/mL (SIGMA chemical company, St.Louis, MO) be dissolved in HBSS (Alcon Laboratories, Fort Worth, TX) in, treatment temperature is 30 ℃, and the solution pH value is 7.0.
After above-mentioned two steps processing, with hetero stroma of cornea place PBS (Invitrogen lifetechnologies, Carlbad, CA) in, 100 rev/mins of rinsings 48 hours, treatment temperature is 4 ℃, and the solution pH value is 7.0, and the cornea after the processing is processed 12 hours in pure glycerin again, dehydration back corneal transparency, referring to Fig. 1 b, Fig. 2 b, Fig. 3 b, be transferred to the medium-term and long-term preservation of 85% glycerite, for using.
Embodiment 2 takes off the preparation of cell Cornu Bovis seu Bubali membrane matrix
In qualified feed lot, select the healthy donors cattle, with after the penthiobarbital pin intramuscular injection anesthesia, win the buphthalmos ball, prune after the eyeball surface affiliated group and eyeball to be soaked in 5% povidone iodine 3 minutes, then with 500 ml physiological salines flushings, be transported to laboratory under 4 degrees centigrade.
Operating microscope lower edge limbus of corneae is cut the full-shape film, scrape off epithelial layer, tear the cornea posterior elastic membrane off, then above-mentioned hetero stroma of cornea is placed 30ml nonionic detergent, 300 rev/mins of uninterrupted concussions 72 hours, changed solution once in per 12 hours, the nonionic detergent adopts 0.5%Triton X-100 (SIGMAchemical company, St.Louis MO) is dissolved in the deionized water, and adds 25mmol/L EDTA (SIGMA chemical company, St.Louis, MO), treatment temperature is 10 ℃, and the solution pH value is 7.0.
Take out the hetero stroma of cornea after the above-mentioned processing, (TX) flushing was 0.5 hour for Alcon Laboratories, FortWorth, and the residual solution on eccysis surface places nuclease solution then, with 300 rev/mins of uninterrupted concussions 1 hour with HBSS.Nuclease solution adopt RNaseA 1mg/mL and DNaseI 10mg/mL (SIGMA chemical company, St.Louis, MO) be dissolved in HBSS (Alcon Laboratories, Fort Worth, TX) in, treatment temperature is 37 ℃, the solution pH value is 7.0.
After handling through above-mentioned two steps, with hetero stroma of cornea place PBS (Invitrogen lifetechnologies, Carlbad, CA) in 100 rev/mins of abundant rinsings 48 hours, treatment temperature is 4 ℃, the solution pH value is 7.0.Cornea after the processing is again through pure glycerin processed 24 hours, and dehydration back corneal transparency is transferred to that 85% glycerite is medium-term and long-term to be preserved, for use.
Embodiment 3 new zealand rabbit therapeutic corneal transplantation experiments
Acellular Cornu Bovis seu Bubali membrane matrix with the preparation of embodiment 2 methods is donor material (to call donor in the following text), selecting healthy new zealand rabbit is receptor bound therapeutic Penetrating Keratoplasty, after rabbit usefulness penthiobarbital intramuscular injection success general anesthesia, the conventional PVP iodine sterilization of art eye, drape, eyelid left by eye speculum, do the inferior retcus anchor suture, donor's cornea with normal saline flushing after in substrate face up and place on the cornea pad, with 7.5 millimeters trepans after recipient cornea central authorities are drilled into the anterior chamber, inject viscoelastic agent in the place ahead, wipe out recipient cornea and prepare plant bed along boring edge with corneal scissors, get and plant sheet and place on the plant bed, with 10-0 nylon wire (Alcon Laboratories, Fort Worth TX) makes 16 pin interrupted sutures.Art finishes removes rectus suture and eye speculum, and (SantanPharmaceutics, Osaka Japan) are coated with eye with the tarivid eye ointment.Postoperative gives once a day, and the tarivid eye ointment is coated with eye.Observe conjunctival congestion, epithelization is finished and situation such as rejection generation.Transplant back discovery conjunctival congestion and disappear after 3 days, the epithelization postoperative was finished in 4 days, and no rejection takes place in 3 months observation period.
Embodiment 4 new zealand rabbit lamellar cornea transplantation experiments
The acellular porcine cornea substrate for preparing with embodiment 1 method is donor material (to call donor in the following text), selecting healthy new zealand rabbit is the receptor bound lamellar keratoplasty, after the penthiobarbital intramuscular injection success general anesthesia, the conventional povidone iodine sterilization of art eye, drape, eyelid left by eye speculum, does the inferior retcus anchor suture, donor's cornea faces up with substrate in normal saline flushing is after 10 minutes and places on the cornea pad, drills through donor with 7.75 millimeters trepans and plants sheet.After recipient cornea central authorities are drilled into about 2/3 cornea degree of depth, separate flaggy with 7.5 millimeters trepans with cornea lamellar blade.Get and plant sheet and place plant bed, (Alcon Laboratories, Fort Worth TX) make 16 pin interrupted sutures with the 10-0 nylon wire.Art finishes removes rectus suture and eye speculum, and the tarivid eye ointment is coated with eye.Postoperative gives once a day, and the tarivid eye ointment is coated with eye.Observation conjunctival congestion, epithelization are finished indexs such as situation and the change of angle transparency.Transplant back discovery conjunctival congestion and disappear after 3 days, epithelization was finished in 4 days, and no rejection takes place in 3 months observation period, and corneal transparency is referring to Fig. 4, Fig. 5.
Need not further to elaborate, believe and adopt the disclosed content in front, those skilled in the art can use the present invention to greatest extent.Therefore, the embodiment of front is interpreted as only illustrating, but not limits the scope of the invention by any way.
The partial reference document that the present invention relates to
1.Whitcher,John?P.,Srinivasan,M.and?Upadhyay,Madan?P.Corneal?blindness:a?globalperspective.Bull?World?Health?Organ,2001,vol.79,no.3,p.214-221.ISSN?0042-9686.
2.Hicks?C,Crawford?G,Chirila?T,et?al.Development?and?clinical?assessment?of?an?artificialcornea.Prog?Retin?Eye?Res?2000;19(2):149-70.
3.Chirila?TV.An?overview?of?the?development?of?artificial?corneas?with?porous?skirts?and?theuse?of?PHEMA?for?such?an?application.Biomaterials?2001;22(24):3311-7.
4.Trinkaus-Randall?V,Nugent?MA.Biological?response?to?a?synthetic?cornea.J?Control?Release1998;53(1-3):205-14.
5.Minami?Y,Sugihara?H,Oono?S.Reconstruction?of?cornea?in?three-dimensional?collagen?gelmatrix?culture.Invest?Ophthalmol?Vis?Sci?1993;34(7):2316-24.
6.Griffith?M,Osborne?R,Munger?R,et?al.Functional?human?corneal?equivalents?constructedfrom?cell?lines.Science?1999;286(5447):2169-72.
7.Kampmeier?J,Radt?B,Bimgruber?R,Brinkmann?R.Thermal?and?biomechanical?parameters?ofporcine?cornea.Cornea?2000;19(3):355-63.
8.Ross?JR,Howell?DN,Sanfilippo?FP.Characteristics?of?corneal?xenograft?rejection?in?adiscordant?species?combination.Invest?Ophthalmol?Vis?Sci?1993;34(8):2469-76.
9.Amano?S,Shimomura?N,Kaji?Y,et?al.Antigenicity?of?porcine?cornea?as?xenograft.Curr?EyeRes?2003;26(6):313-8.
10.Grauss?RW,Hazekamp?MG,van?Vliet?S,et?al.Decellularization?of?rat?aortic?valve?allograftsreduces?leaflet?destruction?and?extracellular?matrix?remodeling.J?Thorac?Cardiovasc?Surg2003;126(6):2003-10.
11.Srivastava?A,Jennings?LJ,Hanumadass?M,et?al.?Xenogeneic?acellular?dermal?matrix?as?adermal?substitute?in?rats.J?Burn?Care?Rehabil?1999;20(5):382-90.
12.Cartmell?JS,Dunn?MG.Effect?of?chemical?treatments?on?tendon?cellularity?and?mechanicalproperties.J?Biomed?Mater?Res?2000;49(1):134-40.
13.Atala?A.Experimental?and?clinical?experience?with?tissue?engineering?techniques?forurethral?reconstruction.Urol?Clin?North?Am.2002?May;29(2):485-92,ix.
Claims (10)
1. an acellular hetero stroma of cornea is a material with the animal corneal, it is characterized in that: provide a kind of acellular hetero stroma of cornea.
2. the preparation method of acellular hetero stroma of cornea according to claim 1 is characterized in that: be achieved through the following technical solutions:
(1) obtains the fresh animal corneal stroma: win animal eyeball, prune and to be soaked in after the eyeball surface affiliated group in 5% povidone iodine 3 minutes, wash with 500 ml physiological salines then, cut the full-shape film along limbus of corneae, tear corneal epithelium and posterior elastic membrane off, obtain animal corneal substrate;
(2) processing of eluting keratocyte film component and removal nucleic acid:
1. the animal corneal substrate that obtains is placed ion-type or nonionic detergent solution, the vibration of constant temperature shaking table was changed 1 time in the every 6-12 of detergent solution hour, and temperature keeps 25 ℃-37 ℃, pH value 6-8, and elution time 24-72 hour,
2. the corneal stroma reuse nuclease solution rinsing through 1. handling, temperature keeps 27 ℃-37 ℃, pH value 6-8,
3. the corneal stroma through 2. handling obtains acellular hetero stroma of cornea with balanced salt solution flushing 48-72 hour;
(3) processed: acellular hetero stroma of cornea is carried out 12-24 hour processed with pure glycerin;
(4) preserve: the acellular hetero stroma of cornea after will dewatering places 85% glycerite to preserve, and is standby.
3. the preparation method of acellular hetero stroma of cornea according to claim 2 can be put the animal eyeball of winning earlier-70 ℃ of stored frozen, the acellular hetero stroma of cornea of refabrication after the normal temperature unfreezing.
4. according to the preparation method of the described acellular hetero stroma of cornea of claim 2-3, the solution of detergent is selected hypotonic solution, and the solution osmotic pressure is 0mosm~299mosm.
5. according to the preparation method of the described acellular hetero stroma of cornea of claim 2-3, ionic detergent preferably sodium dodecyl sulfate (SDS), the preferred bent ketone X100 of nonionic detergent (Triton X-100), the suitable concentration of sodium lauryl sulphate is between 0.05% to 0.1%, and the suitable concentration of bent ketone X100 is between 0.1% to 4%.
6. the preparation method of acellular hetero stroma of cornea according to claim 5 adds collagenase inhibitors in the detergent solution, and suitable concentration is at 1-25mmol/L, and collagenase inhibitors is selected diethylamine tetraacethyl (EDTA).
7. the preparation method of acellular hetero stroma of cornea according to claim 2, nuclease is selected excision enzyme or restriction endonuclease, excision enzyme is selected ribonuclease A (RNaseA) and deoxyribonuclease I (DNaseI), restriction endonuclease can be selected EcoR I and Hind III etc., the concentration range of ribonuclease A be 0.02mg/ml to 1mg/ml, the concentration range of deoxyribonuclease I is that 0.2mg/ml is to 10mg/ml.
8. the preparation method of acellular hetero stroma of cornea according to claim 2 is washed used balanced salt solution preferably phosphoric acid balanced salt solution (PBS).
9. according to the described acellular hetero stroma of cornea of claim 1-8, the application in the artificial cornea's of organizational project structure.
10. according to the described acellular hetero stroma of cornea of claim 1-8, the application in the medical material of preparation corneal transplantation, cornea refractive surgery.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CNB2004100181075A CN100333702C (en) | 2004-04-28 | 2004-04-28 | Exogenous cornea substrate without cells and its preparation method and use |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CNB2004100181075A CN100333702C (en) | 2004-04-28 | 2004-04-28 | Exogenous cornea substrate without cells and its preparation method and use |
Publications (2)
Publication Number | Publication Date |
---|---|
CN1579342A true CN1579342A (en) | 2005-02-16 |
CN100333702C CN100333702C (en) | 2007-08-29 |
Family
ID=34581744
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CNB2004100181075A Expired - Fee Related CN100333702C (en) | 2004-04-28 | 2004-04-28 | Exogenous cornea substrate without cells and its preparation method and use |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN100333702C (en) |
Cited By (27)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007071169A1 (en) * | 2005-12-20 | 2007-06-28 | Zhiguang (Gz) Biotech Co., Ltd | Biological artificial cornea and method of making |
CN100386061C (en) * | 2005-05-17 | 2008-05-07 | 浙江大学医学院附属邵逸夫医院 | Low antigen hetero stroma of cornea treated by frozen, and its prepn. method |
WO2008131639A1 (en) * | 2007-04-25 | 2008-11-06 | Zhongshan Ophthalmic Center, Sun Yat-Sen University | A cell-removing cornea substrate and a method of preparation thereof |
US7674289B2 (en) | 2005-07-29 | 2010-03-09 | Grandhope Biotech Co., Ltd. | Biological artificial ligament and method of making |
CN101947144A (en) * | 2010-09-29 | 2011-01-19 | 厦门大学 | Ply tissue engineering corneal frame and manufacturing method and application thereof |
CN101274106B (en) * | 2008-03-24 | 2011-11-09 | 中山大学中山眼科中心 | Method for preparing acellular matrix |
US8100970B2 (en) | 2005-12-20 | 2012-01-24 | Grandhope Biotech Co., Ltd. | Biological surgical patch and method of making |
CN101433478B (en) * | 2007-11-14 | 2012-05-23 | 上海交通大学医学院附属第九人民医院 | Whole layer biological cornea as well as construction method and use thereof |
US8197500B2 (en) | 2005-08-04 | 2012-06-12 | Grandhope Biotech Co., Ltd. | Biological membrane-carrying aneurysm clip |
CN102599991A (en) * | 2012-03-20 | 2012-07-25 | 中山大学中山眼科中心 | Application of beautifying cornea lens |
US8292799B2 (en) | 2005-07-29 | 2012-10-23 | Grandhope Biotech Co., Ltd. | Biological artificial blood vessel and method of making |
US8366770B2 (en) | 2005-12-20 | 2013-02-05 | Grandhope Biotech Co. Ltd. | Biological artificial nerve guide and method of making |
CN104001215A (en) * | 2014-06-13 | 2014-08-27 | 深圳艾尼尔角膜工程有限公司 | Acellular corneal stroma and preparing method for acellular corneal stroma |
CN104001217A (en) * | 2014-06-13 | 2014-08-27 | 深圳艾尼尔角膜工程有限公司 | Decellularized method for cornea |
CN104288837A (en) * | 2014-09-11 | 2015-01-21 | 杨淑珍 | Acellular dermal matrix and preparation method and application thereof |
CN104645416A (en) * | 2015-01-26 | 2015-05-27 | 中国海洋大学 | In vitro construction method for tissue engineering human corneal stroma |
CN104665956A (en) * | 2015-02-12 | 2015-06-03 | 厦门大开医疗器械有限公司 | Preparation method for artificial corneas |
WO2015188664A1 (en) * | 2014-06-13 | 2015-12-17 | 深圳艾尼尔角膜工程有限公司 | Acellular corneal method, acellular corneal stroma and preparation method thereof |
CN106421908A (en) * | 2016-07-01 | 2017-02-22 | 山东省眼科研究所 | Method for preparing decellularized cornea |
CN106563163A (en) * | 2016-10-25 | 2017-04-19 | 浙江大学 | Electrostatic layer-by-layer self assembling technology modified bioengineering porcine cornea and production method thereof |
CN106614519A (en) * | 2016-11-24 | 2017-05-10 | 山东省眼科研究所 | Decellularized corneal limbus protective liquid and preparation method of decellularized corneal limbus |
CN106729999A (en) * | 2016-11-24 | 2017-05-31 | 山东省眼科研究所 | A kind of method for building tissue engineering comea edge |
CN107412748A (en) * | 2015-09-09 | 2017-12-01 | 北京大学第医院 | A kind of isolated rabbit kerectasis model and construction device based on II Collagenase Types |
CN109444395A (en) * | 2018-10-31 | 2019-03-08 | 清华大学深圳研究生院 | A kind of living body on-spot study inducing tissue regeneration mechanism platform and its application method |
CN109475663A (en) * | 2016-12-16 | 2019-03-15 | 厦门大开生物科技有限公司 | A kind of preparation method of de- cell porcine cornea and its de- cell plate layer cornea and usage |
CN113368313A (en) * | 2021-06-10 | 2021-09-10 | 吾奇生物医疗科技(江苏)有限公司 | Preparation method of biological membrane, product and application thereof |
CN115006594A (en) * | 2022-06-26 | 2022-09-06 | 中国海洋大学 | Preparation method and application of mechanically-enhanced acellular porcine corneal posterior lamella carrier support |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5827641A (en) * | 1992-11-13 | 1998-10-27 | Parenteau; Nancy L. | In vitro cornea equivalent model |
WO2001056379A1 (en) * | 2000-02-07 | 2001-08-09 | The Schepens Eye Research Institute, Inc. | A method of quieting a corneal inflammatory response |
KR20040024859A (en) * | 2001-07-25 | 2004-03-22 | 가부시키가이샤 재팬 티슈 엔지니어링 | Laminates for treating cornea and process for producing the same |
CN1326502C (en) * | 2003-08-07 | 2007-07-18 | 中山大学中山眼科中心 | Artificial tissue engineeing biological cornea |
-
2004
- 2004-04-28 CN CNB2004100181075A patent/CN100333702C/en not_active Expired - Fee Related
Cited By (37)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN100386061C (en) * | 2005-05-17 | 2008-05-07 | 浙江大学医学院附属邵逸夫医院 | Low antigen hetero stroma of cornea treated by frozen, and its prepn. method |
US7824447B2 (en) | 2005-07-29 | 2010-11-02 | Grandhope Biotech Co. Ltd. | Biological artificial ligament and method of making |
US8292799B2 (en) | 2005-07-29 | 2012-10-23 | Grandhope Biotech Co., Ltd. | Biological artificial blood vessel and method of making |
US7674289B2 (en) | 2005-07-29 | 2010-03-09 | Grandhope Biotech Co., Ltd. | Biological artificial ligament and method of making |
US8197500B2 (en) | 2005-08-04 | 2012-06-12 | Grandhope Biotech Co., Ltd. | Biological membrane-carrying aneurysm clip |
US8100970B2 (en) | 2005-12-20 | 2012-01-24 | Grandhope Biotech Co., Ltd. | Biological surgical patch and method of making |
US8366770B2 (en) | 2005-12-20 | 2013-02-05 | Grandhope Biotech Co. Ltd. | Biological artificial nerve guide and method of making |
WO2007071169A1 (en) * | 2005-12-20 | 2007-06-28 | Zhiguang (Gz) Biotech Co., Ltd | Biological artificial cornea and method of making |
CN1985778B (en) * | 2005-12-20 | 2010-10-13 | 广东冠昊生物科技股份有限公司 | Artificial biological cornea |
WO2008131639A1 (en) * | 2007-04-25 | 2008-11-06 | Zhongshan Ophthalmic Center, Sun Yat-Sen University | A cell-removing cornea substrate and a method of preparation thereof |
CN101433478B (en) * | 2007-11-14 | 2012-05-23 | 上海交通大学医学院附属第九人民医院 | Whole layer biological cornea as well as construction method and use thereof |
CN101274106B (en) * | 2008-03-24 | 2011-11-09 | 中山大学中山眼科中心 | Method for preparing acellular matrix |
CN101947144B (en) * | 2010-09-29 | 2012-07-04 | 厦门大学 | Ply tissue engineering corneal frame and manufacturing method and application thereof |
CN101947144A (en) * | 2010-09-29 | 2011-01-19 | 厦门大学 | Ply tissue engineering corneal frame and manufacturing method and application thereof |
CN102599991A (en) * | 2012-03-20 | 2012-07-25 | 中山大学中山眼科中心 | Application of beautifying cornea lens |
CN104001215A (en) * | 2014-06-13 | 2014-08-27 | 深圳艾尼尔角膜工程有限公司 | Acellular corneal stroma and preparing method for acellular corneal stroma |
CN104001217A (en) * | 2014-06-13 | 2014-08-27 | 深圳艾尼尔角膜工程有限公司 | Decellularized method for cornea |
WO2015188664A1 (en) * | 2014-06-13 | 2015-12-17 | 深圳艾尼尔角膜工程有限公司 | Acellular corneal method, acellular corneal stroma and preparation method thereof |
CN104288837A (en) * | 2014-09-11 | 2015-01-21 | 杨淑珍 | Acellular dermal matrix and preparation method and application thereof |
CN104645416A (en) * | 2015-01-26 | 2015-05-27 | 中国海洋大学 | In vitro construction method for tissue engineering human corneal stroma |
CN104665956A (en) * | 2015-02-12 | 2015-06-03 | 厦门大开医疗器械有限公司 | Preparation method for artificial corneas |
CN104665956B (en) * | 2015-02-12 | 2019-04-26 | 厦门大开医疗器械有限公司 | A kind of preparation method of artificial cornea |
CN107412748A (en) * | 2015-09-09 | 2017-12-01 | 北京大学第医院 | A kind of isolated rabbit kerectasis model and construction device based on II Collagenase Types |
CN106421908A (en) * | 2016-07-01 | 2017-02-22 | 山东省眼科研究所 | Method for preparing decellularized cornea |
CN106421908B (en) * | 2016-07-01 | 2018-01-16 | 深圳艾尼尔角膜工程有限公司 | A kind of preparation method of de- cell cornea |
CN106563163A (en) * | 2016-10-25 | 2017-04-19 | 浙江大学 | Electrostatic layer-by-layer self assembling technology modified bioengineering porcine cornea and production method thereof |
CN106563163B (en) * | 2016-10-25 | 2023-02-17 | 浙江大学 | Bioengineering pig cornea modified by electrostatic layer-by-layer self-assembly technology and preparation method |
CN106614519B (en) * | 2016-11-24 | 2019-11-19 | 山东省眼科研究所 | A kind of preparation method of de- cell corneal limbus protection liquid and de- cell corneal limbus |
CN106614519A (en) * | 2016-11-24 | 2017-05-10 | 山东省眼科研究所 | Decellularized corneal limbus protective liquid and preparation method of decellularized corneal limbus |
CN106729999A (en) * | 2016-11-24 | 2017-05-31 | 山东省眼科研究所 | A kind of method for building tissue engineering comea edge |
CN106729999B (en) * | 2016-11-24 | 2019-11-19 | 山东省眼科研究所 | A method of building tissue engineering comea edge |
CN109475663A (en) * | 2016-12-16 | 2019-03-15 | 厦门大开生物科技有限公司 | A kind of preparation method of de- cell porcine cornea and its de- cell plate layer cornea and usage |
CN109475663B (en) * | 2016-12-16 | 2021-11-26 | 厦门大开生物科技有限公司 | Preparation method of acellular porcine cornea, acellular lamellar cornea and use method of acellular lamellar cornea |
CN109444395A (en) * | 2018-10-31 | 2019-03-08 | 清华大学深圳研究生院 | A kind of living body on-spot study inducing tissue regeneration mechanism platform and its application method |
CN113368313A (en) * | 2021-06-10 | 2021-09-10 | 吾奇生物医疗科技(江苏)有限公司 | Preparation method of biological membrane, product and application thereof |
CN113368313B (en) * | 2021-06-10 | 2022-06-03 | 吾奇生物医疗科技(江苏)有限公司 | Preparation method of biological membrane, product and application thereof |
CN115006594A (en) * | 2022-06-26 | 2022-09-06 | 中国海洋大学 | Preparation method and application of mechanically-enhanced acellular porcine corneal posterior lamella carrier support |
Also Published As
Publication number | Publication date |
---|---|
CN100333702C (en) | 2007-08-29 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN100333702C (en) | Exogenous cornea substrate without cells and its preparation method and use | |
CN101274106B (en) | Method for preparing acellular matrix | |
CA2252758C (en) | Grafts made from amniotic membrane; methods of separating, preserving, and using such grafts in surgeries | |
Fernández-Pérez et al. | Decellularization and recellularization of cornea: Progress towards a donor alternative | |
CN101947144B (en) | Ply tissue engineering corneal frame and manufacturing method and application thereof | |
CN101985051A (en) | Acellular cornea or acellular corneal stroma, preparation method and application thereof | |
KR20150127247A (en) | Method for decellularization of tissue grafts | |
CA2491528A1 (en) | Decellularized tissue | |
CN101917932A (en) | Method for processing porcine cornea for decellularization | |
CN1898377A (en) | Human corneal endothelial cells and methods of obtaining and culturing cells for corneal cell transplantation | |
WO2004078225A1 (en) | Amnion-origin medical material and method of preparing the same | |
JP2007509643A (en) | Methods and compositions for growth of corneal endothelium and related cells on biopolymers and creation of artificial corneal grafts | |
CN1311072C (en) | Preparation method of dried active amnion | |
CN1775304A (en) | Method for preparing artificial biological valve with biological activity | |
CN105013013A (en) | Preparation method of skin ulcer repairing matrix | |
WO2010139209A1 (en) | Kit used for obtaining whole liver scaffold and method for obtaining whole liver scaffold | |
CN104001217B (en) | Decellularized method for cornea | |
CN108888804B (en) | Soft tissue repair material and preparation method thereof | |
CN109069263B (en) | Porcine cornea acellular method, acellular cornea and lamellar dry cornea using method | |
CN100484497C (en) | A method for preparing bioactivity possessed artificial cornea | |
KR101859654B1 (en) | Cell scaffold including collagen and decellularized extraceulluar matrix and artificial cornea sheet | |
CN100386061C (en) | Low antigen hetero stroma of cornea treated by frozen, and its prepn. method | |
CN1893988A (en) | Methods and compositions for growing corneal endothelial and related cells on biopolymers and creation of artifical corneal transplants | |
CN108939161B (en) | A kind of humanization activity goes the preparation method of cell corneal stroma stent | |
JP7321152B2 (en) | Elastin reduction to allow recellularization of cartilage grafts |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C14 | Grant of patent or utility model | ||
GR01 | Patent grant | ||
C17 | Cessation of patent right | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20070829 Termination date: 20130428 |