CN100340309C - Biological induction type active artificial cornea, and application method therefor - Google Patents
Biological induction type active artificial cornea, and application method therefor Download PDFInfo
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- CN100340309C CN100340309C CNB021153167A CN02115316A CN100340309C CN 100340309 C CN100340309 C CN 100340309C CN B021153167 A CNB021153167 A CN B021153167A CN 02115316 A CN02115316 A CN 02115316A CN 100340309 C CN100340309 C CN 100340309C
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Abstract
The present invention provides a biologic induction type active artificial cornea which comprises the components of the following weight percentage: 0.5% to 3% of chitosan, 0.5% to 3% of collagen, 0.1% to 0.5% of hyaluronic acid and 93.5% to 98.8% of water. The active artificial cornea is helpful to stimulate the concentration speed of self growth factors and can firstly integrate cornea growth factors on the materials. The present invention also relates to a use method of the biologic induction type active artificial cornea, which comprises the concrete step that the biologic induction type active artificial cornea is directly transplanted into a human eye. The characteristic of the biologic induction type active artificial cornea is similar to the characteristic of the cornea the human eye. The biologic induction type active artificial cornea has the advantages of good compatibility with eye tissue, forming processing adaptation, simple manufacturing process, easy long-period storage and long-distance transportation, and good social and economic benefit. The use method of the artificial cornea has the advantages of simplicity, convenience, safety, reliability and good action effect. The present invention also opens a way for the researches of other kinds of biologic induction type active artificial tissue, and has significant scientific significance.
Description
(1) technical field
The present invention relates to artificial cornea's technology, particularly a kind of biological induction type active artificial cornea and using method thereof.
(2) background technology
Keratopathy is the present principal element of eye blinding, and global patient has 1,000 ten thousand people approximately, China 3,000,000 corneal blindness patients that also have an appointment.But because the scarcity of donor source, the allogeneic keratoplasty that China can carry out every year only is about 1000 examples, and a large amount of patients can not get curing because of the shortage of human cornea, and this is a global long-term unsolved problem.Because the no blood vessel characteristic of eye immune privilege phenomenon and cornea, keratoplasty is the highest operation of success rate in body tissue's organ transplantation, and therefore, artificial cornea and transplanting thereof are better than the transplanting of other organizational project organs and tissue.Present existing artificial cornea has two kinds of nonactive artificial cornea and the epigamic tissue engineering artificial corneals of inanimate object, the artificial cornea that nonactive artificial cornea utilizes synthetic material such as methyl methacrylate (MMA) etc. to make, when carrying out the human implantation, though can make people's vision recovers to some extent, but field range is minimum, synthetic material is difficult to merge with enclosing near the eyes to organize mutually, foreign body sensation is arranged after the implantation, corneal solution, leakage of aqueous humor appear easily, the artificial cornea deviates from and complication such as endophthalmitis, brings very big misery to the patient.And common notional tissue engineering artificial corneal is under the condition of external reinforcement, carries out keratocyte and cultivates, and the cell of In vitro culture just makes it biologically active after growth on the timbering material; Though the artificial cornea that this method is produced may make moderate progress aspect the histocompatibility with enclosing near the eyes, but because it needs the multiple keratocyte of In vitro culture, and the approaches and methods of acquisition of keratocyte at present and cultivation is relatively more difficult and immature, the preservation after keratocyte is cultivated and the also existing problems of breeding that go down to posterity, whole process is complicated, expense is high, and the immunologic rejection effect of allosome keratocyte in migration process also is a great problem that faces.
(3) summary of the invention
The objective of the invention is to overcome the shortcoming of prior art, but provide a kind of and people ocular tissue have excellent biological compatibility, need not be at external cell culture, easy to use, functional and long preservation and long-distance transporting, the biological induction type active artificial cornea of being convenient to carry out large-scale production and realizing industrialization of carrying out.
Another object of the present invention is to provide the using method and the technology of above-mentioned biological induction type active artificial cornea.
Purpose of the present invention is achieved through the following technical solutions: this biological induction type active artificial cornea comprises following component---
Chitosan 0.5%～3%
Collagen 0.5%～3%
Hyaluronic acid 0.1%～0.5%
Water 93.5%～98.8%
Above ratio is weight percentage.
Except that above-mentioned component, for helping stimulate speed from the enrichment of the bulk-growth factor, this biological induction type active artificial cornea also can be integrated with the keratocyte somatomedin as becoming very thin somatomedin (FGF), epithelical cell growth factor (EGF) and cell transformation somatomedin (TGF-β
1), consumption is 3～100ng/ml, the carbodiimide with 0.01%～0.2% (EDC) cross-linking agent is crosslinked, forms the biological induction type active artificial cornea of band somatomedin.
The method for preparing above-mentioned biological induction type active artificial cornea comprises the steps: that (1) is dissolved in acid solution with chitosan, and collagen and hyaluronic acid are water-soluble; (2) mix each component; (3) mixed liquor is injected mould and oven dry; (4) be soaked in water behind cleaning, drying resulting product and make it reach the suction balance.
Behind each component mix homogeneously of step 2, can add the keratocyte somatomedin.
The using method of above-mentioned biological induction type active artificial cornea is: directly this biological induction type active artificial cornea is implanted into human eye, before transplanting, need carry out the cultivation of external keratocyte; After being implanted into human eye, this biological induction type active artificial cornea is organized mutually with cornea and is merged, induce the enrichment of multiple keratocyte somatomedin, stimulate keratocyte growth and breeding thereon, the degraded synchronized development of the growth of keratocyte and artificial cornea's material, final whole artificial cornea's length of a film becomes cornea tissue.
Particularly, the using method of above-mentioned biological induction type active artificial cornea is soaked biological induction type active artificial cornea 12～48 hours at sterile distilled water before comprising the steps: to transplant in advance, make it reach the suction balance, reuse medicining liquid dipping 2～3 days, carry out the preceding aseptic sterilization of art, transplant according to clinical requirement then.
Described disinfectant solution is the mixed liquor of normal saline and gentamycin sulfate.
Action principle of the present invention is: people's cornea tissue mainly is made of epithelial cell, fibroblast, endotheliocyte and collagen fiber and mucopolysaccharide, collagen protein accounts for 75% of cornea dry weight, can from animal, extract in a large number, the source is abundant and the charge is small, therefore adopts the host material of collagen as this artificial CF.
The collagen that from animal, extracts, though similar to human collagen on forming, variation has taken place in its overall structure in leaching process, the collagen after the purification has lost intensity, as the backing substrate material, is subjected to the effect of collagenase in vivo, very easily degraded; In order to remedy these weakness of collagen, strengthen intensity and its degradation rate in vivo of control of collagen, take to add the method for chitosan; Chitosan is a kind of natural biological aminopolysaccharide, to the mucopolysaccharide in the matter between keratocyte similar structure is arranged, good film-forming property, intensity height, appearance transparent, in body not by degraded by collagenase, can be degraded by lysozyme lentamente, thereby can regulate the degradation rate of backing substrate material, it is crosslinked also can to use cross-linking agent that material is carried out simultaneously, the control degradation rate.
Hyaluronic acid belongs to mucopolysaccharide; it is to constitute outside the keratocyte and the main component of intercellular matrix; have viscoelasticity and macromole hydrability; can promote growth and breeding with the pilot angle theca cell; network structure corneal form and the keeping of normal physiological state that forms that combine with collagen has important function; by with the combining of corneal endothelium; form the protecting film on cornea tissue surface; the damage that the radical pair keratocyte causes in the control volume; the hydroxyl that contains in the mucopolysaccharide composition on a large amount of electronegative carboxyls and eye surface in the hyaluronan molecule is easy to and the surperficial affinity of eye by hydrogen bonded.
The cultivation of somatomedin corneal cell, growth and breeding reach the great influence that is combined with extracellular and intercellular matrix, the cooperative effect between the different keratocyte somatomedin and the growth and breeding of consumption corneal cell can be regulated and control, wherein fibroblast growth factor (FGF), transforming growth factor (TGF-β
1), and the growth and breeding of epidermal growth factor (EGF) corneal cell, intercellular normal fusion and facilitation is all arranged with combining of substrate.
The present invention has following advantage with respect to prior art: (1) biological induction type active artificial cornea of the present invention and ocular tissue have the good compatibility, merge mutually with the cornea cell, induce the enrichment of multiple keratocyte somatomedin, stimulate keratocyte growth and breeding thereon; Before corneal transplantation and repairing, need not carry out the cultivation of external keratocyte, thereby exempted the complex process of In vitro culture keratocyte and transplanted the immunological rejection cause by the allosome keratocyte, and the various complication that produce therefrom, so use not only simple and convenient, and as safe as a house reliable, action effect is better; (2) this biological induction type active artificial cornea has optics similar to eye cornea and mechanical characteristic, can carry out various machine-shapings according to relevant requirements, can be used as the comparatively ideal succedaneum of eye cornea; The optics mechanical index of the biological induction type active artificial cornea of having made is similar to people's cornea, as follows: material light transmittance: 90～96%; Material refractive index: 1.368～1.382 (eye cornea is 1.377); Mechanical strength and eye cornea are similar, wherein hygrometric state tensile strength: 4Mpa～8.6MPa; Moisture content: 55～75%; Elongation at break: 300%～350%; (3) this biological induction type active artificial cornea relatively is fit to processing and forming, and manufacturing process is simple, handling ease is convenient, so can prepare use in large quantity; (4) this biological induction type active artificial cornea is easy to long preservation and long-distance transporting, and is convenient to carry out large-scale production and realizes industrialization; (5) invention of this biological induction type active artificial cornea, not only important facilitation is arranged for people ' s health, can produce important society and economy benefit, but also opened up road for the research of other biological induction type active artificial organ, as adopt similar biotic induce method can realize the direct implantation of other artificial organ, thereby has important scientific meaning.
(4) specific embodiment
Below in conjunction with embodiment the present invention is done further concrete description, but embodiments of the present invention are not limited thereto.
Embodiment 1
The ratio of each component of this biological induction type active artificial cornea is as follows:
Chitosan 2%
Collagen 2.5%
Hyaluronic acid 0.3%
Water 95.1%
Above ratio is weight percentage.
The process for preparing this biological induction type active artificial cornea with above-mentioned each component comprises the steps: that (1) takes by weighing each component by above-mentioned consumption, and chitosan is dissolved in hydrochloric acid solution, and collagen and hyaluronic acid is water-soluble; (2) chitosan salt acid solution and collagen and hyaluronic aqueous solution were at room temperature stirred 1.5 hours, make the abundant mix homogeneously of each component; (3) the carbodiimide cross-linking agent of adding 0.1% in the mixed liquor of mix homogeneously, 90ng/ml becomes very thin somatomedin, and 35ng/ml epithelical cell growth factor and 60ng/ml cell transformation somatomedin fully mix half an hour again; (4) mixed liquor is injected ready-formed cornea mould, fill with and take out after putting it into drying in oven behind the cornea mould; (5) with the cornea goods of clear water soaking and washing oven dry back gained removing residual impurity, and make it in water, place 24～48 hours to reach the suction balance, take out then and promptly make this biological induction type active artificial cornea.
The using method of this biological induction type active artificial cornea is: the biological induction type active artificial cornea material is soaked 24h at sterile distilled water in advance, make it reach the suction balance, cut the sequin that is cut into thickness 0.3～0.5mm with 8mm diameter trepan, with normal saline and gentamycin sulfate mixed liquid dipping 2～3 days, carry out aseptic sterilization before the art.Transplant according to clinical requirement then, enclose near the eyes with iodine tincture sterilization receptor during transplanting, splash into 1～2 ***e eye drop anaesthetic at its ophthalmic and carry out local anesthesia; Postoperative subconjunctival injection dexamethasone and gentamycin sulfate are coated with tetracycline cortisone ointment in the conjunctival sac; Postoperative drips and be coated with gentamycin sulfate eye drop and tetracycline cortisone ointment every day, or subconjunctival injection dexamethasone and gentamycin sulfate, keeps for 6～8 weeks, the back drug withdrawals of 8 weeks, and be 1～12 month observing time, as finding rejection, i.e. repeated drug taking.
Embodiment 2
The ratio of each component of this biological induction type active artificial cornea is as follows:
Chitosan 1%
Collagen 3%
Hyaluronic acid 0.2%
Water 95.65%
Above ratio is weight percentage.
The process for preparing this biological induction type active artificial cornea with above-mentioned each component comprises the steps: that (1) takes by weighing each component by above-mentioned consumption, and chitosan is dissolved in acetum, and collagen and hyaluronic acid is water-soluble; (2) chitosan-acetic acid solution and collagen and hyaluronic aqueous solution were at room temperature stirred 2 hours, make the abundant mix homogeneously of each component; (3) the carbodiimide cross-linking agent of adding 0.15% in the mixed liquor of mix homogeneously, 5ng/ml becomes very thin somatomedin, and 20ng/ml epithelical cell growth factor and 10ng/ml cell transformation somatomedin fully mix half an hour again; (4) mixed liquor is injected ready-formed cornea mould, fill with and take out after putting it into drying in oven behind the cornea mould; (5) with the cornea goods of clear water soaking and washing oven dry back gained removing residual impurity, and make it in water, place 24～48 hours to reach the suction balance, take out then and promptly make this biological induction type active artificial cornea.
The using method of this biological induction type active artificial cornea is with embodiment 1.
Embodiment 3
The ratio of each component of this biological induction type active artificial cornea is as follows:
Chitosan 0.5%
Collagen 1.5%
Hyaluronic acid 0.3%
Water 97.7%
Above ratio is weight percentage.
The process for preparing this biological induction type active artificial cornea with above-mentioned each component comprises the steps: that (1) takes by weighing each component by above-mentioned consumption, and chitosan is dissolved in acetum, and collagen and hyaluronic acid is water-soluble; (2) chitosan-acetic acid solution and collagen and hyaluronic aqueous solution were at room temperature stirred 2 hours, make the abundant mix homogeneously of each component; (3) mixed liquor is injected ready-formed cornea mould, fill with and take out after putting it into drying in oven behind the cornea mould; (4) with the cornea goods of clear water soaking and washing oven dry back gained removing residual impurity, and make it in water, place 24～48 hours to reach the suction balance, take out then and promptly make this biological induction type active artificial cornea.
The using method of this biological induction type active artificial cornea is with embodiment 1.
Embodiment 4
The ratio of each component of this biological induction type active artificial cornea is as follows:
Chitosan 1.5%
Collagen 2%
Hyaluronic acid 0.1%
Water 96.4%
Above ratio is weight percentage.
The process for preparing this biological induction type active artificial cornea with above-mentioned each component comprises the steps: that (1) takes by weighing each component by above-mentioned consumption, and chitosan is dissolved in acetum, and collagen and hyaluronic acid is water-soluble; (2) chitosan-acetic acid solution and collagen and hyaluronic aqueous solution were at room temperature stirred 2 hours, make the abundant mix homogeneously of each component; (3) mixed liquor is injected ready-formed cornea mould, fill with and take out after putting it into drying in oven behind the cornea mould; (4) with the cornea goods of clear water soaking and washing oven dry back gained removing residual impurity, and make it in water, place 24～48 hours to reach the suction balance, take out then and promptly make this biological induction type active artificial cornea.
The using method of this biological induction type active artificial cornea is with embodiment 1.
The transplanting result who observes the various embodiments described above is as can be known: the biological induction type artificial cornea material of implantation can be compatible with cornea, and is bonding between cornea and the material, with cornea mutually adhesion the visible keratocyte in material place and grow to material internal; Two months degradation rates in vivo of material are about 30%.Taking out dyeing after 90 days makes the result that the pathology section observes and show under optical microscope: cornea structure is normal, the NIP cell, epithelium does not have and thickens and attenuation, essential layer does not have edema, interior skin structure is normal, material has substantially all grown up to cornea tissue, with former cornea tissue good transition is arranged, and does not see difference.
Claims (3)
1, a kind of biological induction type active artificial cornea comprises following component:
Chitosan 0.5%～3%
Collagen 0.5%～3%
Hyaluronic acid 0.1%～0.5%
Water 93.5%～98.8%
Above ratio is weight percentage.
2, according to the described biological induction type active artificial cornea of claim 1, it is characterized in that: being integrated with to stimulate in the inductor keratocyte breeding of growing into, the keratocyte somatomedin that makes it need not carry out the cell in vitro cultivation before implantation.
3, according to the described biological induction type active artificial cornea of claim 2, it is characterized in that: described keratocyte somatomedin is a fibroblast growth factor (FGF), epithelical cell growth factor and cell transformation somatomedin, consumption is respectively 3～100ng/ml, and is crosslinked with 0.01%～0.2% carbodiimide cross-linking agent.
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| Application Number | Priority Date | Filing Date | Title |
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| CNB021153167A CN100340309C (en) | 2002-05-31 | 2002-05-31 | Biological induction type active artificial cornea, and application method therefor |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021153167A CN100340309C (en) | 2002-05-31 | 2002-05-31 | Biological induction type active artificial cornea, and application method therefor |
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| CN1461658A CN1461658A (en) | 2003-12-17 |
| CN100340309C true CN100340309C (en) | 2007-10-03 |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100512889C (en) * | 2006-12-08 | 2009-07-15 | 华南理工大学 | Process of making cornea histoengineering support in bionic structure |
| CN102600495A (en) * | 2011-01-19 | 2012-07-25 | 北京博恩康生物科技有限公司 | Absorbable hemostatic composition and preparation method thereof |
| CN103272268B (en) * | 2013-05-16 | 2015-04-22 | 华南理工大学 | Antibacterial cornea repairing material and preparation method thereof |
| CN115006595B (en) * | 2022-06-28 | 2024-02-06 | 济南金泉生物科技有限公司 | Composite high-performance decellularized cornea and preparation method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH05220213A (en) * | 1992-02-13 | 1993-08-31 | Nippon Soda Co Ltd | Chitosan derivative-containing artificial blood vessel |
| US5522888A (en) * | 1986-10-16 | 1996-06-04 | Cbs Lens, A California General Partnership | Collagen-hydrogel for promoting epithelial cell growth and regeneration of the stroma |
| JPH09173362A (en) * | 1995-12-25 | 1997-07-08 | Menicon Co Ltd | Artificial skin |
| JPH1076000A (en) * | 1996-09-06 | 1998-03-24 | Agency Of Ind Science & Technol | Cell adhesive and hyperplastic medical treatment article |
-
2002
- 2002-05-31 CN CNB021153167A patent/CN100340309C/en not_active Expired - Lifetime
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5522888A (en) * | 1986-10-16 | 1996-06-04 | Cbs Lens, A California General Partnership | Collagen-hydrogel for promoting epithelial cell growth and regeneration of the stroma |
| JPH05220213A (en) * | 1992-02-13 | 1993-08-31 | Nippon Soda Co Ltd | Chitosan derivative-containing artificial blood vessel |
| JPH09173362A (en) * | 1995-12-25 | 1997-07-08 | Menicon Co Ltd | Artificial skin |
| JPH1076000A (en) * | 1996-09-06 | 1998-03-24 | Agency Of Ind Science & Technol | Cell adhesive and hyperplastic medical treatment article |
Non-Patent Citations (1)
| Title |
|---|
| 天然大分子及其生物材料在组织工程中的应用 李沁华，现代康复，第5卷第6期 2001 * |
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