CN100484497C - A method for preparing bioactivity possessed artificial cornea - Google Patents
A method for preparing bioactivity possessed artificial cornea Download PDFInfo
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- CN100484497C CN100484497C CNB2005100428123A CN200510042812A CN100484497C CN 100484497 C CN100484497 C CN 100484497C CN B2005100428123 A CNB2005100428123 A CN B2005100428123A CN 200510042812 A CN200510042812 A CN 200510042812A CN 100484497 C CN100484497 C CN 100484497C
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Abstract
The invention relates to a method for preparing biologically active artificial cornea. It takes animal cornea basic material as raw material, promoting cornea growth or /and preventing partial lesion by enzyme slaking, repeated unfreezing, washing, and irradiating with 60Co. It employs physical and chemical method to remove cell component and soluble protein which will cause immune response, and retains external base material construction, adds multifunctional component which is favor for cornea cell growth or /and preventing partial lesion, freeze dries and stores it. The tensile strength and diopter of prepared artificial cornea are similar to that of normal cornea, it can prevent lesion and dissolution after being implanted in, and promote cornea epithelium regeneration and collagen synthesis; the biocompatibility is good, no obvious immune rejection reaction and no toxic to cell, and can be used to treat various eye injury.
Description
Technical field
The invention belongs to the artificial organ technical field of biomaterial, be specifically related to a kind of biologically active artificial cornea's preparation method.
Background technology
Diseases such as cornea inflammation, wound, chemical burn, birth defect often cause corneal injury, cause visual deterioration even blinding, the method of corneal transplantation is the most effective clinically solution, but the source famine of allosome cornea donor, and also need to join type before transplanting, restricted carrying out of corneal graft greatly, the patient of a large amount of corneal injuries can not be got timely medical treatment, finally cause losing one's sight.In China, annual patient because of the corneal injury blinding has 300,000 approximately, and the patient that can accept corneal transplantation only has 2000 many cases.At some in particular cases, for example chemical burn, eye pemphigoid, Stevens-Johnson syndrome, corneal graft rejection etc. repeatedly, the success rate of corneal transplantation is just very low.Therefore press for a kind of cornea substitute for transplanting clinically, this class substitute has following requirement: (1) has good biocompatibility and histocompatibility; (2) have suitable mesh-structuredly, help growing into of cell; (3) have certain machinability, can be processed into different diopters according to actual needs, implanting finally can be transparent fully; (4) comprise the functional component that promotes the keratocyte growth and stop the local patholoic change development.Though at present more to the research of artificial cornea's biomaterial, all have certain defective, can not satisfy requirement of actual application.
Amniotic membrane is to use more a kind of cornea cladding material at present clinically, have complete basement membrane and extracellular matrix, biocompatibility is higher, shortcoming is that amniotic membrane is thinner relatively, thickness only is 20～100 μ m, be only applicable to the shallow degree damage of treatment eye table, for the degree of depth or the damaged unsatisfactory curative effect of corneal stroma.
Comparatively speaking, the heterogenic cornea source is abundant, has and organizational structure like the human cornea matrix phase, and thickness, the diopter of transplanting the back cornea remain unchanged, and sensory nerve can be grown into, and consciousness can be recovered; Yet wherein contained cell component and stromatin composition all will cause heterogenic cornea to transplant the back produces intensive immunological rejection, causes graft failure.If can remove the immunogenicity of heterogenic cornea, then can avoid the appearance of this problem, solve the insufficient problem of clinical cornea donor.The method of using always in studying at present is that the method for employing detergent and nuclease is removed the cell component in the heterogenic cornea, Chinese patent (200410018107.5) " a kind of acellular hetero stroma of cornea and preparation method and purposes " as the Yao Yufeng application, its preparation method is: with the fresh animal cornea that obtains, with ion-type or nonionic detergent eluting keratocyte film component, re-use the nucleic acid enzyme process and remove nucleic acid; Standby with preserving after the acellular hetero stroma of cornea processed.The problem that exists is because its heterogenous animal cornea that adopts is handled through taking off cell, has only removed endothelial cell and stromal cell, does not consider the biological incompatible immunological rejection that causes of residual xenogenesis stromatin composition; Used simultaneously multiple detergent in preparation process, its inevitable residual meeting pair cell toxigenicity influences clinical therapeutic efficacy; And this invention cornea after handling do not contain the function ingredients that some suppress local patholoic changes development and help the keratocyte growth, is difficult to obtain ideal repairing effect.
Summary of the invention
At the deficiencies in the prior art, the objective of the invention is to propose a kind of artificial cornea's of biologically active preparation method, make the artificial cornea of preparation have the hot strength similar, diopter to normal cornea, can stop pathological development and corneal solution after implanting the affected part, promote regeneration of corneal epithelium and substrate collagen synthetic, reduce new vessels and generate; Have excellent biological compatibility simultaneously, do not have tangible immunological rejection, the pair cell avirulence can be treated various ocular injurys.
For achieving the above object, the preparation of the artificial cornea of the present invention includes the pre-treatment step of animal corneal, the present invention be with pretreated cornea adopt enzymic digestion, multigelation, washing,
60Co irradiation, the growth of compound promotion keratocyte are or/and stop the step preparation of the function ingredients of local patholoic change development.Its concrete grammar is operated according to following steps:
(1) enzymic digestion: pretreated animal corneal is placed 1000～8000U/ml protein enzyme solution digestion 30 minutes～12 hours, to remove cornea epidermis cell and endothelial cell, the length of digestion time depends on the digestive environments temperature, and temperature is low, and then digestion time is long; Reuse distilled water rinsing cornea behind the residual protein of flush away, continues to soak with distilled water, makes the abundant swelling of stromal cell in the cornea.
(2) multigelation: the cornea after the swelling was placed liquid nitrogen at least 30 minutes, guarantee that the inside and outside temperature of cornea reaches consistent; Take out cornea and be placed under the room temperature and thaw naturally, so multigelation is 2～5 times, makes the cell disintegrate of breaking fully; With distilled water rinsing cornea, flush away is residual;
(3) washing: the cornea after the rinsing is placed the solution dissolved cell that contains surfactant (as NaTDC, cetomacrogol 1000 etc.), concussion washing 6～12 hours, the lipid in the dissolving cornea, protein etc.; With distilled water rinsing cornea, purpose is with dissolved cell component and the thorough eluting of surfactant;
(4)
60Co irradiation: the cornea lyophilizing after will washing, the cornea after the lyophilizing is used
60Co radiation, irradiation accumulated dose are 5～20kGY, and purpose is sterilization on the one hand; Can make residual stromatin degeneration inactivation on the one hand, to remove the antigen of immunological rejection that heterogenic cornea is caused; Cornea behind the irradiation can store at low temperatures;
(5) cornea after the radiation treatment can also be according to the recipient cornea degree of impairment, compound promotion keratocyte growth is or/and stop the function ingredients of local patholoic change development, it can be a kind of or several in epithelical cell growth factor, platelet-derived growth factor, transforming growth factor, basic fibroblast growth factor, vascular endothelial cell growth factor, nerve growth factor, collagenase inhibitors, antibiotic, the cysteine, make it have anti-infection ability, and the destruction that can suppress the wound surface collagen fiber, promote the healing of cornea wound surface.Composite methods can adopt forging bone described in (Chinese patent 98125671.6 Instructions Page 2s) document and BMP composite methods or other prior aries to carry out.Can adopt lyophilizing to store.
(6) optics processing: as required, can adopt cornea ablation cutter or laser to carry out Surface Machining, make it have certain diopter, to satisfy the optics requirement of receptor eye to the artificial cornea of preparation.
The present invention is prepared has the treatment that active artificial cornea can be used for various corneal injuries, deposits under 4 ℃ of environment and can preserve more than 1 year.
The present invention is used to repair artificial cornea's the preparation method of human cornea damage and the artificial cornea who obtains by its method compares with product with prior art, has the following advantages:
1) preparation method of the present invention has that cost is low, method is easy, the advantage of easy operating.
2) artificial cornea of the present invention preparation can be applicable to the corneal ulcer that reasons such as inflammation, ulcer, chemical burn cause or the corneal graft of perforation of cornea, has certain elasticity, toughness, and its shape size and thickness are easy to change.
3) artificial cornea of the present invention preparation is treated to the platinum sponge shape through lyophilizing, has mesh-structuredly, can promote normally adhering to and growing of cell, implants and can be reconstructed by recipient cell gradually, finally can be transparent fully.
4) artificial cornea of the present invention's preparation has the hot strength similar to human cornea, diopter, is easy to process according to the demand of receptor.
5) the present invention is owing to mainly use physical method to remove antigenic component in preparation process, compare the use that has significantly reduced chemical substance with prior art processes, especially do not adopt pair cell that bigger toxic triton x-100 is arranged, it is residual to have reduced chemical substance, improved the biocompatibility of the cornea that obtains, be beneficial to by body and accept; And the surfactant that uses of the present invention (as NaTDC etc.) has easy eluting, the characteristics of less residue.
6) but the artificial cornea of the present invention preparation by compound promotion keratocyte growth and the function ingredients that stops the local patholoic change development after implanting the sick wound surface that decreases infection, stop ulcer to develop and corneal solution, promote the synthetic and epithelium regeneration of substrate collagen, reduce new vessels and generate, finally can form the structure similar to normal cornea.
7) artificial cornea of the present invention preparation by hypisotonic solution swelling, multigelation, surfactant washing,
60Behind the Co irradiation, remove the cell component and the stromatin activity of heterogenic cornea, greatly reduced the immunological rejection that xenotransplantation causes.
8) artificial cornea of the present invention preparation characteristics that also have easy to use, wide material sources and be easy to store have excellent curative to various common ocular injurys, are a kind of cornea repair materials preferably.
Accompanying drawing and explanation thereof
The photo of artificial cornea's outward appearance of Fig. 1, the present invention's preparation, spongiosis is white in color.
The photo of artificial cornea's scanning electron microscope of Fig. 2, the present invention's preparation, visible anterior corneal surface is vesicular texture.
Fig. 3, rabbit corneal ulcer photo, visible cornea is damaged, and there is purulent secretion the part.
Fig. 4, the artificial cornea who uses the present invention to prepare repair the postoperative photo of rabbit corneal ulcer, as seen plant sheet and plant bed and sew up closely, are recessed plate-like.
The lagophthalmos portion photo in February behind the artificial keratoplasty of Fig. 5, the present invention's preparation, visible corneal transparence obviously strengthens.
The lagophthalmos portion photo in April behind the artificial keratoplasty of Fig. 6, the present invention's preparation, corneal transparency under the visible slit lamp, curvature nature, thickness uniformity.
The specific embodiment
Below will the present invention is described further by embodiment:
It is that material carries out pretreatment that the present invention can select the cornea of cattle, pig, horse, sheep for use, and the animal corneal with the peel-away removal surrounding tissue soaks in normal saline, cuts to desired thickness after making its swelling.
1) enzymic digestion: trypsin solution (25 ℃) digestion at room temperature that pretreated Oculus sus domestica cornea is placed 4000U/ml 1 hour.With distilled water rinsing cornea, the protease that flush away is residual; Again cornea is placed distilled water to soak 4 hours, make the abundant swelling of intracorneal stromal cell;
2) multigelation: place liquid nitrogen (-196 ℃) to soak 1 hour the cornea after the swelling; The taking-up cornea is placed under the room temperature and thaws naturally, and so multigelation is 4 times, with distilled water rinsing cornea;
3) washing: the cornea after the freeze thawing is placed the deoxycholic acid sodium solution of 40g/L concentration, and (15 ℃) concussion washing is 6 hours under the room temperature, with distilled water rinsing cornea;
4)
60Co irradiation: the cornea after will washing places vacuum freeze drier to carry out lyophilizing, and the cornea after the lyophilizing is used
60Co radiation, irradiation accumulated dose are 16kGY, adopt 4kGY/ minute dose irradiation 4 minutes;
5) corneal film after the radiation treatment is placed immersion taking-up lyophilizing storage after 6 hours in the buffer solution that contains 5 μ g/ml epidermal growth factors, 5 μ g/ml basic fibroblast growth factors and 5% (m/v) cysteine (destruction that can suppress cornea wound surface collagen fiber promotes ulcer healing).Attached Fig. 1 and 2 is the artificial cornea's of preparation photo.
6) optics processing: use cornea ablation cutter or laser that its surface is processed to prepared artificial cornea, make it have certain diopter, to satisfy the optics requirement of receptor eye.
Animal experiment adopts 2 monthly age new zealand rabbits (Fig. 3) of eye table ulcer, and 2% pentobarbital sodium lumbar injection 30mg/kg anaesthetizes to the soft breathing of extremity steadily, and anatomic microscope with 6mm trepan excision pathological changes cornea, dissects the degree of depth and meets the pathological changes degree of depth down.With the smooth sutured on the plant bed (Fig. 4) that places of 6.5mm artificial cornea, operation method is identical with conventional lamellar cornea transplant operation with postoperative care with 10-0 nylon wire.The result shows, one week of postoperative, the cornea of implanting begins translucent, postoperative February, cornea recovers transparent (Fig. 5), and 4 months gross examination of skeletal muscle corneas of postoperative definition is good, corneal transparency under the slit lamp, the curvature nature, honest and kind uniformity (Fig. 6) is not seen cornea tissue edema and inflammatory reaction on every side in repair process.
Claims (3)
1, a kind of artificial cornea's of biologically active preparation method includes the pre-treatment step of animal corneal, and compound promotion keratocyte growth is characterized in that or/and stop the step of the function ingredients of local patholoic change development, concrete operations according to the following steps:
(1) enzymic digestion: pretreated animal corneal is placed 1000～8000U/ml protein enzyme solution digestion 30 minutes～12 hours, reuse distilled water rinsing cornea, the distilled water immersion cornea makes its abundant swelling;
(2) multigelation: the cornea after the swelling was placed liquid nitrogen at least 30 minutes, and the taking-up cornea is placed under the room temperature and thaws naturally, and so multigelation is 2～5 times, with distilled water rinsing cornea;
(3) washing: the cornea after the rinsing is placed the solution dissolved cell that contains surfactant, and concussion washing 6～12 hours is with distilled water rinsing cornea;
(4)
60Co irradiation: the cornea lyophilizing after will washing, the cornea after the lyophilizing is used
60Co radiation, irradiation accumulated dose are 5～20kGY.
2, preparation method according to claim 1, it is characterized in that, or/and stop in the step of function ingredients of local patholoic change development, described function ingredients is a kind of or several in epithelical cell growth factor, platelet-derived growth factor, transforming growth factor, basic fibroblast growth factor, vascular endothelial cell growth factor, nerve growth factor, collagenase inhibitors, antibiotic, the cysteine in described compound promotion keratocyte growth.
3, preparation method according to claim 1 is characterized in that, adopts cornea ablation cutter or laser to carry out surface optical processing to the cornea after the complex function component.
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101947144B (en) * | 2010-09-29 | 2012-07-04 | 厦门大学 | Ply tissue engineering corneal frame and manufacturing method and application thereof |
| CN101985051A (en) * | 2010-10-21 | 2011-03-16 | 暨南大学 | Acellular cornea or acellular corneal stroma, preparation method and application thereof |
| CN104083803B (en) * | 2013-04-01 | 2016-04-27 | 陕西佰傲再生医学有限公司 | A kind of biomembrane for Ocular surface healing and preparation method thereof |
| CN103750922B (en) | 2013-12-31 | 2016-07-13 | 金仕生物科技（常熟）有限公司 | The method preparing Cardiac valve prosthesis leaflet |
| CN104001214B (en) * | 2014-05-28 | 2015-06-17 | 青岛中皓生物工程有限公司 | Lamellar corneal stroma bracket as well as preparation method and application thereof |
| CN106860914B (en) * | 2017-01-19 | 2020-02-18 | 浙江大学 | Method for obtaining calcium phosphate/extracellular matrix film through cell sheet layer |
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| 分子生物学技术进展对角膜移植的影响. 李绍伟，谢立信.中华眼科杂志，第39卷第11期. 2003 * |
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| 异种角膜移植术临床研究. 马生虎，陈建宏，金秉义，苗林.眼科新进展，第20卷第1期. 2000 |
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