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Modified acellular corneal stroma and modification method thereof

A corneal stroma, decellularization technology, used in pharmaceutical formulations, pharmaceutical science, tissue regeneration, etc., can solve problems such as uncontrollable, dry eye, residual cross-linking agent by-products, etc., to improve transparency and elastic modulus , good transparency and moisture content, improve the effect of biological stability

Active Publication Date: 2016-04-20
欧亚科(广东)生命科学有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Specifically: physical method, mainly through the introduction of ionic or amphiphilic cross-linking agents, with the help of van der Waals force, ionic bonds, hydrogen bonds, hydrophilic and hydrophobic forces, etc. to achieve modification. The advantages of this method are simple operation and easy removal of residues. However, the disadvantages of this method are poor stability, low and uncontrollable cross-linking efficiency, and easy degradation and loss. At the same time, the potential and hydrophilicity and hydrophobicity of the corneal stroma are changed, which may cause immune rejection; chemical and enzymatic methods, through the introduction of cross-linking Linking agents (such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), N-hydroxysuccinimide (NHS), 1,1-carbonyldiimidazole, genipin , oxidized sodium alginate, glutaraldehyde, etc.), enzymes (transglutaminase, horseradish peroxidase, tyrosinase, lysyl oxidase, horseradish peroxidase, etc.) or light (ultraviolet , X-rays, etc.) catalysis to achieve cross-linking of collagen in the corneal stroma. The advantage is that the covalent bonds formed are very stable, efficient, and the conditions are controllable. The disadvantage is that the cross-linking is irreversible under physiological conditions and destroys the dynamics of collagen in the cornea. The hexagonal lattice structure causes secondary damage to the decellularized corneal stroma, and this method also has the problem of residual cross-linking agents, enzymes and by-products
[0006] The above modification methods also have a common problem: they only try to repair the collagen network structure of the acellular corneal stroma, and the design does not involve the restoration of corneal physiological functions, which cannot meet the clinical needs of rapid recovery of corneal transparency.
From the perspective of the physiological function of the cornea, the bound water and free water contained in the cornea are closely related to its transparency. The two are mainly obtained through hydrogen bonds and the hexagonal lattice arrangement of collagen fibers. The recovery rate of the decellularized cornea will be delayed after transplantation, and there is a hidden danger of dry eye syndrome after transplantation in clinical practice.

Method used

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  • Modified acellular corneal stroma and modification method thereof
  • Modified acellular corneal stroma and modification method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] The modified acellular corneal stroma of this example was prepared by the following method:

[0047] (1) Pretreatment of decellularized corneal stroma

[0048] The porcine decellularized corneal stroma was immersed in 5 mL of normal saline, and stood overnight in a refrigerator at 4 °C. The transparent part was cut out with an ophthalmic drill, rinsed with deionized water three times, and then frozen in a -20 °C refrigerator for 4 hours, freeze-dried, and placed at room temperature. Store in a desiccator for later use.

[0049] (2) Graft modification of decellularized corneal stroma

[0050] Soak the dried porcine acellular corneal stroma in step (1) in 5 mL (6 mg / mL) of N-hydroxysuccinimide (NHS) in phosphate buffered saline solution (PBS, pH=7.25), and sonicate at 4°C for 2 h ( 300W, 40Hz). Add 1 mL (13 mg / mL) of 1-(3-dimethylamino Propyl)-3-ethylcarbodiimide (EDC) was activated at 4°C for 2h, then added to porcine decellularized corneal stroma soaked in NHS, plac...

Embodiment 2

[0073] The modified acellular corneal stroma of this example was prepared by the following method:

[0074] (1) Pretreatment of decellularized corneal stroma

[0075] The porcine decellularized corneal stroma was immersed in 5 mL of normal saline, left in a refrigerator at 4°C overnight, and the transparent part was cut out with an ophthalmic drill, rinsed three times with deionized water, and stored in a refrigerator at 4°C for later use.

[0076] (2) Graft modification of decellularized corneal stroma

[0077] With embodiment 1.

[0078] (3) Post-processing of grafted modified acellular corneal stroma

[0079] With embodiment 1.

[0080] The modified acellular corneal stroma prepared in this example was subjected to a rehydration experiment (the test method was the same as in Example 1), and the test results showed that after 10 minutes of rehydration, the modified acellular corneal stroma obtained in this example had a water content of 53%; after 24 hours of rehydration...

Embodiment 3

[0082] The modified acellular corneal stroma of this example was prepared by the following method:

[0083] (1) Pretreatment of decellularized corneal stroma

[0084] Take rabbit decellularized corneal stroma and immerse it in 10mL of ultrapure water, let it stand overnight at room temperature, use an ophthalmic drill to cut out the transparent part, and store it in a 4°C refrigerator for later use.

[0085] (2) Graft modification of decellularized corneal stroma

[0086] Soak the rabbit decellularized corneal stroma treated in step (1) in 5mL (0.08g / mL) of N-hydroxysuccinimide (NHS) in PBS (pH=7.25) solution, sonicate at 4°C for 2h (500W, 40Hz). In 4 mL (10 μg / mL) of glutamic acid in borax-sodium hydroxide buffer solution (pH = 9), add rabbit decellularized corneal stroma soaked in NHS, place on a shaker at 80 rad / min, and react at 0 ° C for 24 h , and then use cobalt 60 for photocatalysis (25KyG, 5min).

[0087] (3) Post-processing of grafted modified acellular corneal s...

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Abstract

The invention belongs to the field of tissue engineering, and particularly relates to modified acellular corneal stroma and a modification method thereof. The modification method comprises the following steps: (1) carrying out pretreatment on acellular corneal stroma; (2) reacting with the pretreated acellular corneal stroma by amino acid, and carrying out graft modification on the acellular corneal stroma; and (3) carrying out post treatment on the acellular corneal stroma subjected to graft modification, and removing the residual material in the graft modification process to obtain the modified acellular corneal stroma. The modified acellular corneal stroma has good mechanical property and biocompatibility, also has excellent rehydration water-retaining properties, can quickly restore the transparency after being implanted, and can be applied to tissue engineering research, clinical corneal transplantation and the like.

Description

technical field [0001] The invention relates to the field of tissue engineering, in particular to a modified acellular corneal stroma and a modification method thereof. Background technique [0002] The cornea is located on the front wall of the eyeball and is a transparent tissue that mainly performs barrier and refractive functions. Because the cornea is in direct contact with the outside world, it is extremely vulnerable to mechanical trauma, thermal burns, acid and alkali erosion, etc., which can cause corneal diseases, and even blindness in severe cases. Corneal transplantation is the only clinical treatment available, but limited by the severe shortage of corneal donors, many corneal patients have lost hope while waiting. With the development of tissue engineering and regenerative medicine, it is possible to construct corneal substitutes with tissue functions similar to those of the cornea in vitro, which is expected to solve the problem of insufficient corneal donors...

Claims

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Application Information

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IPC IPC(8): A61L27/36A61L27/50
CPCA61L27/36A61L27/3683A61L27/3687A61L27/50A61L2400/18A61L2430/16
Inventor 武征赵巨鹏张建华
Owner 欧亚科(广东)生命科学有限公司
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