CN1973911A - Process of making cornea histoengineering support in bionic structure - Google Patents
Process of making cornea histoengineering support in bionic structure Download PDFInfo
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- CN1973911A CN1973911A CN 200610124118 CN200610124118A CN1973911A CN 1973911 A CN1973911 A CN 1973911A CN 200610124118 CN200610124118 CN 200610124118 CN 200610124118 A CN200610124118 A CN 200610124118A CN 1973911 A CN1973911 A CN 1973911A
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Abstract
The present invention discloses one kind of cornea histoengineering support in bionic structure and its preparation process. The process includes the following steps: dissolving chitosan and collagen in acid solution separately and mixing the components via vibrating dispersion; pouring the mixture solution into mold and air drying; treating with alkali solution to obtain film and stoving; and water absorption balancing. The cornea histoengineering support is one ordered layered structure similar to human corneal stroma layer structure, and has transmittance of 91-96 %, wet mechanical strength of 3.9-12.7 MPa, water content of 82.5-143 %, performance parameters similar to that of human cornea, and excellent bioactivity and biocompatibility. It may be used in medical field to replace damage cornea tissue, and has simple preparation process. The present invention has excellent application foreground and scientific significance.
Description
Technical field
The present invention relates to a kind of tissue engineering comea technology, particularly relate to cornea histoengineering support of a kind of bionic structure and preparation method thereof.
Background technology
The cornea illness is the commonly encountered diseases and the frequently-occurring disease of ophthalmology, also is one of main causes of blindness, is only second to cataract and occupies second, has nearly 1/3 to be to be caused by keratopathy among the blind patient of eyes in the world today.In some areas of China, corneal blindness occupies first of all kinds of causes of blindness, and at present, the whole nation is because of about 200,000 examples of corneal blindness patient to be performed the operation, and keratoplasty is the extremely important Therapeutic Method that corneal blindness is recovered lost eyesight.But the source of allogeneic cornea donor lacks, limit this Clinical application, these corneal blindness rehabilitation of patients that develop into of artificial cornea's technology have been brought new hope, and the success or not of the performance corneal transplant operation of corneal material has very important influence.
Traditional artificial cornea's material biocompatibility is poor, be difficult to degraded, and cell is difficult to really stick, breed, the nutrition and metabolism material can not in time exchange, and the binding site interface problem more complicated of support and light beam, support and ocular tissue, the incidence rate height of inflammation.Make up the focus that cornea has become the research of ophthalmology educational circles with tissue engineering technique at present.With the three-dimensional In vitro culture of keratocyte on CF, implant ophthalmic then earlier, when cell differentiation became new cornea tissue, also just degraded was complete for support.So its corneal material of seeking not only will have suitable mechanical strength and excellent biological compatibility, optical property and degradability preferably more will be arranged, and catabolite is nontoxic to human body, nonirritant, degradation rate will be complementary with people's cornea tissue growth rate, also performances such as suitable water absorption rate, hydrophilic will be arranged.Searching meets the material of cornea histoengineering support requirement, and prepares function and eye cornea is approaching, and structure can be a difficult problem that faces with the similar bionic-type cornea histoengineering support of eye cornea.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, the preparation method of the cornea histoengineering support of a kind of function and bionic structure is provided.
A kind of preparation method of cornea histoengineering support of bionic structure comprises the steps:
(1) chitosan is dissolved in 0.5%～2% (volume ratio, down together) acetic acid aqueous solution by 10mg/ml concentration (the 10mg chitosan is dissolved in the 1ml acetic acid solution);
(2) the chitosan acetic acid aqueous solution with step (1) mixes with 2～5mg/ml collagenic aqueous solution;
(3) add 0.1～0.5wt% cross-linking agent crosslinking Treatment in the mixed liquor with step (2) gained;
(4) solution with step (3) gained injects natural air drying under the mould room temperature;
(5) aqueous slkali soaking that adds 2～5wt% in the mould of step (4) was taken off film after 20～35 minutes;
(6) goods of baking step (5) soaked in deionized water 2～4 hours before using, and made it reach the suction balance, obtained the cornea histoengineering support of the bionic structure of ordered laminar.
The collagen of described step (2) is the type i collagen of cattle.
Being mixed into of described step (2) stirred under the room temperature 1～2 hour, and supersonic oscillations were removed bubble in 5～10 minutes.
The cross-linking agent of described step (3) is 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxy-succinamide (NHS).
The aqueous slkali of described step (5) is 2%～5% sodium hydroxide solution or ammonia.
The cornea histoengineering support of the bionic structure of method for preparing, cornea histoengineering support internal structure stratiform is orderly, similar with eye cornea hypothallus internal structure, light transmittance is 91～96%, the hygrometric state mechanical strength is 3.9～12.7MPa, moisture content is 82.5%～143%, and performance parameter and eye cornea are approaching, has good biological activity and biocompatibility.
Action principle of the present invention is: eye cornea mainly is divided into epithelial layer, hypothallus and endodermis, hypothallus accounts for 90% of corneal thickness, arranged with the stratiform ordered structure by collagenous fiber bundle, and be rich in mucopolysaccharide, this layer structure makes cornea have the light transmission of height.Cornea histoengineering is that the transparency is good to the requirement of support, and mechanical strength is suitable, and degradation speed is controlled, biocompatibility excellence etc.Chitosan film transparent good, mechanical strength is good, and in human body, can slowly be degraded by lysozyme, biocompatibility is good, and catabolite is to the nontoxic nonirritant of human body, and its molecular structure is similar to the mucopolysaccharide composition of cornea, wide material sources, abundant raw material meets the requirement of cornea histoengineering support and industrialization; The collagen source is abundant, mature preparation process, the biocompatibility excellence, and be the constituent of eye cornea, the compatibility of tropocollagen molecule and chitosan molecule is good, so select collagen and chitosan blend for use, can improve the pliability of chitosan so simultaneously, further improve the biocompatibility of chitosan.Prepared chitosan and collagen composite membrane support can present and the similar layer structure of people's cornea by the mode ordered arrangement of self assembly, reach structural bionical.
The present invention compared with prior art has following advantage:
(1) cornea histoengineering support internal structure stratiform of the present invention is orderly, and is similar with eye cornea hypothallus internal structure, has good biological activity and biocompatibility.
(2) light transmittance of cornea histoengineering support of the present invention can reach 95% (eye cornea is 95%～98%), the hygrometric state mechanical strength is between 3.9～12.7MPa (eye cornea is about 4.0MPa), moisture content 82.5%～143% (eye cornea is 72%～82%), these performance parameters and eye cornea are approaching.
(3) raw material drawing of the present invention's employing is extensive, cheap, and moulding process is simple, and cost is low, helps large-scale production.
(4) the present invention can change the structure and the mechanical strength of timbering material by regulating the content and the technological parameter of each composition of timbering material.
Description of drawings
Fig. 1 is the sem photograph of cornea histoengineering support of the bionic structure of embodiment 1 preparation;
Fig. 2 is the sem photograph of cornea histoengineering support of the bionic structure of embodiment 2 preparation;
Fig. 3 is the sem photograph of cornea histoengineering support of the bionic structure of embodiment 3 preparation;
Fig. 4 is an eye cornea cross section sketch map;
Fig. 5 is that people's corneal limbal epithelial cell is at 72 hours light microscopic photo of embodiment 1 rack surface growth;
Fig. 6 is that people's corneal limbal epithelial cell is at 72 hours light microscopic photo of embodiment 2 rack surfaces growth.
The specific embodiment
The invention will be further described below in conjunction with embodiment; but be pointed out that following examples can not be interpreted as limiting the scope of the invention, those skilled in the art's foregoing according to the present invention still belongs to protection scope of the present invention to some nonessential improvement and the adjustment that the present invention makes.
Embodiment 1
The preparation method of the cornea histoengineering support of bionic structure comprises the steps:
(1) 1% chitosan is dissolved in 0.5% the acetic acid aqueous solution;
(2) be that the aqueous solution 60ml of 0.2% type i collagen mixes with the chitosan acetic acid aqueous solution 70ml of step (1) and quality than concentration; And after stirring 1.5 hours, sonic oscillation was removed bubble in 10 minutes;
(3) add 0.1%～0.2% cross-linking agent crosslinking Treatment in the mixed liquor with step (2) gained, cross-linking agent is EDC and NHS, in 30 minutes cross-linking agent is dropwised, use magnetic stirrer simultaneously, make that cross-linking agent homodisperse, reaction temperature are room temperature, the response time is 2～4 hours;
(4) solution with step (3) gained injects natural air drying under the mould room temperature;
(5) immersion of the sodium hydroxide solution of adding 2% was taken off film after 30 minutes in the mould of step (4);
(6) baking step (5) goods soaked in deionized water 2～4 hours before using, and made it reach the suction balance, obtained the cornea histoengineering support of bionic structure.After testing, the cornea histoengineering support of this bionic structure comprises following component:
Chitosan 37.8%
Collagen 16.2%
Water 46.0%
Each component all is weight percentage.
Because chitosan molecule and tropocollagen molecule have the excellent compatibility, in solution, can reach the homodisperse of molecular level, along with the continuous volatilization of solvent, two kinds of intermolecular self assembly effects make that cornea histoengineering support internal structure stratiform is orderly.Fig. 4 is an eye cornea cross section sketch map; Account for the layer structure of the hypothallus structure of eye cornea thickness about 90% as can be seen, and the layer structure of this rule has guaranteed the light transmission of cornea excellence for rule.Fig. 1 is the sem photograph of cornea histoengineering support of the bionic structure of present embodiment preparation; The cornea histoengineering support of the bionic structure of the present invention's preparation has inner layer structure as can be seen from Figure 1, and this structure and eye cornea hypothallus internal structure are similar, also possess the good optical performance.The cornea histoengineering support performance test situation of the bionic structure of present embodiment preparation sees Table 1.Fig. 5 is the light microscopic photo that people's corneal limbal epithelial cell was grown 72 hours at the present embodiment rack surface, and the adhesion of people's corneal limbal epithelial cell and proliferation experiment result have shown that the cornea histoengineering support that makes has good biological activity and biocompatibility as shown in Figure 5.
Embodiment 2
The preparation method of the cornea histoengineering support of bionic structure comprises the steps:
(1) 1% chitosan is dissolved in 2% the acetic acid aqueous solution;
(2) be that the aqueous solution 60ml of 0.5% type i collagen mixes with the chitosan acetic acid aqueous solution 70ml of step (1) and quality than concentration; And after stirring 1.5 hours, sonic oscillation was removed bubble in 10 minutes;
(3) solution with step (2) gained injects natural air drying under the mould room temperature;
(4) in the mould of step (3), add the ammonia spirit immersion and take off film after 30 minutes;
(5) baking step (4) goods soaked in deionized water 2～4 hours before using, and made it reach the suction balance, obtained the cornea histoengineering support of bionic structure.After testing, the cornea histoengineering support of this bionic structure comprises following component:
Chitosan 18.5%
Collagen 27.8%
Water 53.7%
Each component all is weight percentage.
Fig. 2 is the sem photograph of cornea histoengineering support of the bionic structure of present embodiment preparation; The cornea histoengineering support of the bionic structure of the present invention's preparation has inner layer structure as can be seen from Figure 2, and this structure and eye cornea hypothallus internal structure are similar, also possess the good optical performance.The cornea histoengineering support performance test situation of the bionic structure of present embodiment preparation sees Table 1.Fig. 6 is the light microscopic photo that people's corneal limbal epithelial cell was grown 72 hours at the present embodiment rack surface, and the adhesion of people's corneal limbal epithelial cell and proliferation experiment result have shown that the cornea histoengineering support that makes has good biological activity and biocompatibility as shown in Figure 6.
Embodiment 3
The preparation method of the cornea histoengineering support of bionic structure comprises the steps:
(1) 1% chitosan is dissolved in 1% the acetic acid aqueous solution;
(2) be that the aqueous solution 60ml of 0.25% type i collagen mixes with the chitosan acetic acid aqueous solution 70ml of step (1) and quality than concentration; And after stirring 1.5 hours, sonic oscillation was removed bubble in 10 minutes;
(3) solution with step (2) gained injects natural air drying under the mould room temperature;
(4) the sodium hydroxide aqueous slkali soaking of adding 2% was taken off film after 30 minutes in the mould of step (3);
(5) baking step (4) goods soaked in deionized water 2～4 hours before using, and made it reach the suction balance, obtained the cornea histoengineering support of bionic structure.After testing, the cornea histoengineering support of this bionic structure comprises following component:
Chitosan 16.5%
Collagen 24.7%
Water 58.8%
Each component all is weight percentage.Fig. 3 is the sem photograph of cornea histoengineering support of the bionic structure of present embodiment preparation; The cornea histoengineering support of the bionic structure of the present invention's preparation has inner layer structure as can be seen from Figure 3, and this structure and eye cornea hypothallus internal structure are similar, also possess the good optical performance.The cornea histoengineering support performance test situation of the bionic structure of present embodiment preparation sees Table 1.
Measure function that the various embodiments described above make and bionic structure cornea histoengineering support optics, mechanical index and eye cornea parameter more as shown in Table 1:
Table one
| The CF performance parameter | The support light transmittance | The hygrometric state hot strength | Moisture content | Contact angle |
| Embodiment 1 | 96％ | 8.8MPa | 85％ | 90.8° |
| Embodiment 2 | 93％ | 3.9MPa | 113％ | 76° |
| Embodiment 3 | 91％ | 6.4MPa | 146％ | 78.5° |
| The eye cornea value | 95％～98％ | About 4.0MPa | 72％～82％ | --- |
| Existing patent (CN1382425A) | 92％～96％ | 3.8～4.2MPa | 65％～76％ | --- |
Remarks:
1, the sensing equipment of light transmittance is the Spectrumentlab 22 PC spectrophotometers of Prism Optical Technology Co, has tested the light light transmittance situation of cornea histoengineering support under the visible light condition of different wave length after the suction balance;
2, the hygrometric state hot strength is to adopt day AG-I type electronic universal tester of island proper Tianjin company, and the cornea histoengineering support after the suction balance is tested 25 ℃ of temperature, rate of extension 10mm/min;
3, moisture content is the ratio according to the dry support dry weight of crossing before the moisture content gain in weight of the cornea histoengineering support before and after the suction balance and the balance that absorbs water;
4, contact angle is the contact angle size of the OCA15 type surface contact angle analyzer not moisture CF material of test when dripping 100s of adopting German Dataphysics company.
The data of table one as can be seen, the support performance of this research is compared with prior art (CN1382425A), the printing opacity of support, rate is more approaching, all can satisfy the optical property requirement of tissue engineering comea to support, and before the hygrometric state hot strength of the support of this patent and water content be better than, this had very important meaning to clinical practice, can better satisfy the requirement of clinical operation operation to the mechanics of materials;
In addition, the internal structure of this support presents and the similarly regular layer structure of eye cornea substrate, has reached bionic structure in a way.This structure plays the effect of grating on the one hand, make the light in the same way that sees through the support visible light strengthen mutually, scattered beam is cancelled out each other, guaranteed the light transmittance of support, on the other hand, molecule regularly arranged makes that the mechanical strength of support is higher, can satisfy clinical mechanical property requirements to the tissue engineering comea support.
Claims (6)
1, a kind of preparation method of cornea histoengineering support of bionic structure is characterized in that comprising the steps:
(1) chitosan is dissolved in 0.5%～2% (volume ratio, down together) acetic acid aqueous solution by 10mg/ml concentration (the 10mg chitosan is dissolved in the 1ml acetic acid solution);
(2) the chitosan acetic acid aqueous solution with step (1) mixes with 2～5mg/ml collagenic aqueous solution;
(3) add 0.1～0.5wt% cross-linking agent crosslinking Treatment in the mixed liquor with step (2) gained;
(4) solution with step (3) gained injects natural air drying under the mould room temperature;
(5) aqueous slkali soaking that adds 2～5wt% in the mould of step (4) was taken off film after 20～35 minutes;
(6) goods of baking step (5) soaked in deionized water 2～4 hours before using, and made it reach the suction balance, obtained the cornea histoengineering support of the bionic structure of ordered laminar.
2, the preparation method of the cornea histoengineering support of bionic structure according to claim 1, the collagen that it is characterized in that described step (2) is the type i collagen of cattle.
3, the preparation method of the cornea histoengineering support of bionic structure according to claim 1 it is characterized in that being mixed into of described step (2) stirred under the room temperature 1～2 hour, and supersonic oscillations was removed bubble in 5～10 minutes.
4, the preparation method of the cornea histoengineering support of bionic structure according to claim 1, the cross-linking agent that it is characterized in that described step (3) are 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide and N-hydroxy-succinamides.
5, the preparation method of the cornea histoengineering support of bionic structure according to claim 1, the aqueous slkali that it is characterized in that described step (5) are 2%～5% sodium hydroxide solution or ammonia.
6, by the cornea histoengineering support of the bionic structure of the described method of claim 1 preparation, it is characterized in that cornea histoengineering support internal structure stratiform is orderly, similar with eye cornea hypothallus internal structure, light transmittance is 91～96%, the hygrometric state mechanical strength is 3.9～12.7MPa, moisture content is 82.5%～143%, and performance parameter and eye cornea are approaching, has good biological activity and biocompatibility.
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| CN102908666A (en) * | 2012-10-11 | 2013-02-06 | 天津市赛瑞生物技术有限公司 | Collagen scaffold material for cornea |
| CN104548201A (en) * | 2015-01-22 | 2015-04-29 | 华南理工大学 | Cornea tissue repairing material and preparation method thereof |
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| CN107261208A (en) * | 2017-05-19 | 2017-10-20 | 广州市朴道联信生物科技有限公司 | A kind of preparation method of the cornea repair material of carrying medicament by different level |
| CN109568663A (en) * | 2018-11-27 | 2019-04-05 | 中山大学中山眼科中心 | The preparation method and cornea scaffold of cornea scaffold |
| CN109568663B (en) * | 2018-11-27 | 2021-01-15 | 中山大学中山眼科中心 | Preparation method of corneal stent material and corneal stent material |
| CN110511409A (en) * | 2019-08-19 | 2019-11-29 | 华南理工大学 | A kind of preparation method of collagen-based Cellulose nanocrystal bluk recombination film |
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