CN1253214C - Manufacturing method of canthus membrane cell carrier membrane support capable of biodegradation - Google Patents
Manufacturing method of canthus membrane cell carrier membrane support capable of biodegradation Download PDFInfo
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- CN1253214C CN1253214C CN 02135307 CN02135307A CN1253214C CN 1253214 C CN1253214 C CN 1253214C CN 02135307 CN02135307 CN 02135307 CN 02135307 A CN02135307 A CN 02135307A CN 1253214 C CN1253214 C CN 1253214C
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- chitosan
- formic acid
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- carrier film
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Abstract
The present invention relates to a method for preparing a biodegradable support for a corneal carrier film, which is characterized in that formic acid colloidal solution of chitosan and N-acetyl chitosan is used as raw material, and a film is prepared on a flat plate or in a concave surface. After the film is dried, formic acid and plasticizing agents in the film are removed by alkaline liquor, and then, the film is washed by water, dried, alkalized and crosslinked. The flat film is pressed into the corneal cell attached carrier film support with a specific curvature, and after being dried, the concave film directly becomes the corneal cell attached carrier film support with a specific curvature. The present invention has the advantages that the prepared film support can be biologically degraded by lysozyme in organisms, aminoglucose as a degraded product can be absorbed and utilized by organisms, the transparence of a formed tissue can reach more than 90% of a normal cornea, and no adverse reaction exist.
Description
The present invention relates to a kind of manufacture method of biodegradable canthus membrane cell carrier membrane support.
The cornea transplantation is to treat the blind effective means of cornea now, during operation, normally with the cornea of allosome by operation transplantation to corneal blindness patient's eye, make eyes recover visual performance.But the allosome cornea can only derive from corpse, because the famine of allosome cornea donor causes a large amount of corneal blindness patients to can not get timely treatment, the allosome cornea has certain immunogenicity simultaneously, can cause the failure of operation sometimes.Therefore there is the people to pass through corneal epithelial cell, stromal cell, endotheliocyte are cultivated on request on the carrier film timbering material that cell attaches, behind the tissue to be formed, again with the active structural transplantation of this cell tissue with physiological function pleasing to the eye in, in the hope of reaching the purpose of recovering lost eyesight.(McCulley JP et al.Ophthalmology, 1980 such as McCulley; 87:194-201) once attached carrier as endothelial cell with gelatin film, by cultivating, the rabbit corneal endotheliocyte attaching of cultivating is grown on the gelatin film, and it is implanted in the rabbit auto corneal that removes endodermis, to observe the endotheliocyte physiological function of transplanting and the biological degradability of gelatin film.The result shows, this gelatin film for a long time can not biodegradation in cornea, so make the test can not be successful.(Elizabeth J et al.Tissue Eng.2000 such as Elizabeth; Be carrier 6:30) with collagen sponge membrane with holes, cultivate people's corneal epithelial cell, stromal cell, endotheliocyte respectively, the result proves that cell can attach, growing multiplication on collagen sponge membrane, but the plastidogenetic transparency of organization degree of collagen sponge culture matrix only can reach 50% of normal cornea, so do not reach the requirement of clinical research.In addition, people's amniotic membrane is considered to attach the best carrier of growth, (Koizumi N et al.Cornea, 2000 such as Koizumi as keratocyte at present; 19:65) the personnel selection amniotic membrane attaches the carrier of growth as keratocyte, the rabbit corneal limbal stem cell is planted sheet plant on it and cultivate, and the epithelium posterius cell covered whole amniotic membrane and attached good in 14 days.Rabbit is carried out autotransplantation can reach epithelization, but amniotic membrane often can not be by biodegradation in the body, and produce immunological rejection.
The manufacture method that the purpose of this invention is to provide a kind of biodegradable cornea carrier film support, it can remedy the above-mentioned deficiency of prior art and material.
A kind of manufacture method of biodegradable cornea carrier film support, it is characterized in that the formic acid colloid solution with chitosan and N-acetyl chitosan is raw material, make film on flat board or in the concave surface, after the drying, with formic acid and the plasticizer in the alkali liquor removal film, water washing, dry, make the diaphragm alkalization again, carry out cross-linking reaction, and the keratocyte that dull and stereotyped diaphragm is pressed into specific curvature attached the carrier film support, then directly become the keratocyte that specific curvature is arranged after the concave surface diaphragm drying and attach the carrier film support.
Advantage of the present invention is that the diaphragm support of manufacturing can be by the biodegradations such as lysozyme in the body, and the catabolite glucosamine can be absorbed by body, and the transparency of organization degree of formation can reach more than 90% of normal cornea, and does not have untoward reaction.
Below by embodiment the present invention is described.
Embodiment 1
(1) colloid solution preparation: take by weighing chitosan and N-acetyl chitosan respectively, their weight is respectively 1g and 1.5g, add in the clean triangular flask or other glass containers, stir adding 96.5g formic acid and 1g plasticizer glycerol down, under 8～-10 ℃ of conditions, dissolve, after placement treats that polysaccharide all is dissolved into colloid, adopt 2# or 3# sand core funnel to filter, remove micro-insoluble matter, get transparent limpid colloid solution, leave standstill or the vacuum condition degassing down.The molecular weight ranges of used chitosan and N-acetyl chitosan is 200KD～800KD, and both weight ratios are 1: 0.4～2, and total percentage by weight is 2～3%, and the concentration expressed in percentage by weight of plasticizer is 0.5～5.Plasticizer is glycerol or butanediol or ethylene glycol or propylene glycol.
(2) system film: get clean polystyrene board or engineering plastics plate or glass plate, the adjustment level is measured the colloid solution of 40ml, is poured on 300cm
2On the flat board, make colloid evenly smooth, dry in fume hood.Behind the drying and forming-film, the flat board of band film is put into NaOH or the KOH solution of 0.1～1.0mol/L, in and remaining formic acid and remove plasticizer in the film, the alkali treatment time can be 2～24 hours.Film is taken off, put into the distilled water washing to neutral, dry under the natural conditions, it is standby to get film.
(3) the diaphragm-operated preparation of band curvature: the flat film that will prepare cuts a certain size garden sheet with trepan with eye, be dipped in the NaOH of 40～50% (weight ratios) or the KOH solution and alkalize, the diaphragm of alkalization is sloughed unnecessary alkali liquor, immerse in the cross-linking agent, cross-linking agent can be certain density glutaraldehyde or epoxychloropropane or ethylene glycol bis glycidyl ether or propylene glycol bisglycidyl ether.It is 3～6Kg/cm that diaphragm is placed on 50～90 ℃, pressure
2Down compacting and make keratocyte through cross-linking reaction and attach the carrier film support of punching block with special curvature.
The membrane support made from specific curvature is put into the distilled water washing to neutral, transfer in the ethanol water of 50～60% (weight ratios) further washing organic residue, further wash with tri-distilled water, normal saline, 121 ℃ of high pressure sterilization or sterilizations in 75% ethanol (aqueous solution) down are stored in the aseptic D-hanks liquid standby in water.
Embodiment 2
(1) colloid solution preparation (ditto)
(2) the diaphragm-operated preparation of band curvature: in fume hood, draw the formic acid colloid solution of certain amount of chitosan and N-acetyl chitosan and plasticizer with pipettor, be added in revolvable diameter under the driven by motor and be in the concave surface of polyfluortetraethylene plate of 14mm, open motor, rotary speed is controlled at 600～800 commentaries on classics/min, makes colloid solution form uniform adhesive film, centrifugal drying under the natural conditions in concave surface, treat that the organic solvent volatilization finishes, and forms the membrane support that has specific curvature uniformly.
The diaphragm-operated processing of curvature: the membrane support that takes out exsiccant band curvature, put into NaOH or the KOH solution of 0.1～1.0mol/L, soak neutralization and remove remaining formic acid and plasticizer, place 2hr, taking-up is put into distilled water and washed to pH6, and is crosslinked in the glutaraldehyde water solution of intrusion 0.2%, reuse tri-distilled water, normal saline washing, 121 ℃ of high pressure descend in sterilization or 75% ethanol water to sterilize in water, are stored in the aseptic D-hanks liquid standby.
The present invention attaches the carrier material of growth with natural chitosan and the N-acetylated chitosan sugar that can be degraded by organism as the preparation keratocyte.The keratocyte of preparation cultivate to attach the carrier film support, and its curvature R value is 6.8,7.8 respectively, 8.0mm, and diameter is 10-14mm, wet thickness is 40-80um, transmittance 〉=90%, wet water content are 60-70%, and carrier film support permeability is that the MW2000 material all can be free penetrating.
Claims (1)
- The manufacture method of 1 one kinds of biodegradable cornea carrier film supports, it is characterized in that the formic acid colloid solution with chitosan and N-acetyl chitosan is raw material, make film on flat board or in the concave surface, after the drying, with formic acid and the plasticizer in the alkali liquor removal film, water washing, dry, make the diaphragm alkalization again, carry out cross-linking reaction, and the keratocyte that dull and stereotyped diaphragm is pressed into specific curvature attached the carrier film support, then directly become the keratocyte that specific curvature is arranged after the concave surface diaphragm drying and attach the carrier film support; Described colloid solution is made up of 1g chitosan, 1.5gN-acetyl chitosan, 1g plasticizer and 96.5g formic acid; The molecular weight of described chitosan and N-acetyl chitosan is 200KD-800KD, and described plasticizer is a glycerol; Described specific curvature R value is 6.8,7.8,8.0mm, and diameter is 10-14mm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02135307 CN1253214C (en) | 2002-07-18 | 2002-07-18 | Manufacturing method of canthus membrane cell carrier membrane support capable of biodegradation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02135307 CN1253214C (en) | 2002-07-18 | 2002-07-18 | Manufacturing method of canthus membrane cell carrier membrane support capable of biodegradation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1513563A CN1513563A (en) | 2004-07-21 |
| CN1253214C true CN1253214C (en) | 2006-04-26 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN 02135307 Expired - Fee Related CN1253214C (en) | 2002-07-18 | 2002-07-18 | Manufacturing method of canthus membrane cell carrier membrane support capable of biodegradation |
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| CN (1) | CN1253214C (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100998526B (en) * | 2006-01-10 | 2011-10-05 | 上海组织工程研究与开发中心 | Corneal graft |
| CN101112625B (en) * | 2006-07-24 | 2010-09-01 | 陶鹭 | Chitose shield for treating ceratonosus and method of manufacturing the same |
| CN100512889C (en) * | 2006-12-08 | 2009-07-15 | 华南理工大学 | Process of making cornea histoengineering support in bionic structure |
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2002
- 2002-07-18 CN CN 02135307 patent/CN1253214C/en not_active Expired - Fee Related
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| CN1513563A (en) | 2004-07-21 |
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