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Acellular cornea or acellular corneal stroma, preparation method and application thereof

A corneal stroma, decellularization technology, applied in the field of medical materials, can solve the problems of opaque tensile strength, low light transmittance, long decellularization time, etc., and achieve the effect of retaining transparency, short processing time, and low immunogenicity

Inactive Publication Date: 2011-03-16
JINAN UNIVERSITY
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AI Technical Summary

Problems solved by technology

[0005] The classic tissue engineering cornea is to inoculate the seed cells on the degradable material. Minami et al. cultivated the corneal cells on the collagen gel. Although the three-dimensional corneal analog was obtained, it could not be used for corneal transplantation due to its opacity and weak tensile strength.
Griffith et al. cultured transgenic endothelial and epithelial cells on a matrix based on collagen-chondroitin sulfate, and successfully cultivated functional human-like corneal tissue, but viruses and aldehyde cross-linking are toxic to normal cells, and the strength of the matrix In addition, because collagen is easily digested by collagenase, it is unstable, which in turn affects the optical properties of the cornea and the long-term survival of transplanted cells
Therefore, artificially synthesized corneal biomaterials are still far from ideal, and it is impossible to prepare a matrix structure with similar transparency, strength and biological characteristics as the human cornea.
[0006] Liu Xiaoxia et al. used the trypsin-freezing method to decellularize fresh rabbit corneas. The results showed that the full-thickness rabbit corneas and layered acellular matrix obtained by this method had no cellular components, but the light transmittance was lower than that of fresh corneas and decellularized. It takes a long time to last for 3 days
Xu Yonggen from the Third Affiliated Hospital of Peking University et al. used surfactants, DNase and RNase to decellularize the natural porcine cornea. After dehydration of 90% glycerol for 4 hours, its transparency was close to that of the natural cornea, but the natural corneal stroma was destroyed. Ultrastructure
Since most of the decellularization methods currently used such as mechanical force, ionic and deionized detergents, and enzymatic treatments can affect the transparency of the cornea and destroy the ultrastructure of the cornea

Method used

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  • Acellular cornea or acellular corneal stroma, preparation method and application thereof
  • Acellular cornea or acellular corneal stroma, preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Example 1: Preparation of porcine full-thickness cornea decellularized cornea

[0037] (1) Acquisition of fresh porcine cornea: obtain fresh porcine eyeballs from pig carcasses slaughtered in a certain slaughterhouse, and use phosphate buffered saline (PBS, pH value) containing 2000u / ml of gentamicin after trimming the eyeball surface accessory tissues 7) wash 3 times, and cut out the full-thickness pig cornea along the corneal limbus under the operating microscope, see figure 1 a. figure 2 a. image 3 a: The initial weight of the obtained porcine full-thickness cornea is weighed under the condition of the full-thickness cornea at room temperature of 20°C to 28°C;

[0038] (2) Remove corneal epithelium, endothelium and stromal cells:

[0039] ① Soak the obtained porcine full-thickness cornea in pure water for 0.5 h at room temperature;

[0040] ② Put each piece of pig full-thickness cornea treated in step ① into 10ml 200U / ml phospholipase A 2 solution, placed on a...

Embodiment 2

[0047] Example 2: Preparation of porcine corneal stroma decellularized matrix

[0048] (1) Acquisition of fresh porcine corneal stroma: obtain fresh porcine eyeballs from pig carcasses slaughtered in a certain slaughterhouse, and use phosphate buffered saline (PBS, pH The value is 8) Washing 4 times, cutting the porcine full-thickness cornea along the corneal limbus under the operating microscope; scraping off the corneal epithelium and tearing off the descemet layer from the porcine full-thickness cornea to obtain the porcine corneal stroma; room temperature 20 ° C ~ 28 ° C Next, the obtained porcine corneal stroma was weighed for initial initial weight;

[0049] (2) Remove corneal stromal cells:

[0050] ①At room temperature (20°C-28°C), soak the obtained porcine corneal stroma in pure water for 0.5h;

[0051] ② Put each piece of porcine corneal stroma after step ① into 15ml 300U / ml phospholipase A 2 solution, placed on a constant temperature shaker for 3 hours, the treat...

Embodiment 3

[0055] Example 3: Preparation of bovine full-thickness cornea decellularized cornea

[0056] (1) Acquisition of fresh bovine full-thickness cornea: obtain fresh bovine eyeballs from cattle carcasses slaughtered in a certain slaughterhouse, and use 2000u / ml gentamicin-containing phosphate buffered saline (PBS, The pH value is 7.5) and washed 5 times, and the whole cornea was cut along the limbus under an operating microscope to obtain a bovine full-thickness cornea; at a room temperature of 20°C to 28°C, the initial weight of the obtained bovine full-thickness cornea was weighed;

[0057] (2) Remove corneal epithelium, endothelium and stromal cells:

[0058] ①At room temperature (20°C-28°C), soak the obtained bovine cornea in pure water for 1 hour;

[0059] ② Put each piece of bovine full-thickness cornea treated in step ① in 15ml 2.4U / ml neutral protease Dispase II solution, place it on a constant temperature shaker and digest it for 5 hours with shaking, the treatment temper...

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Abstract

The invention discloses acellular cornea or acellular corneal stroma, a preparation method and application thereof. The method comprises the following steps of: (1) obtaining fresh animal full-thickness cornea or corneal stroma; (2) removing corneal epithelium, corneal endothelium and stroma cells, namely 1, soaking the full-thickness cornea or the corneal stroma in pure water at room temperature; 2, placing the soaked full-thickness cornea or corneal stroma into enzyme solution, digesting with oscillating, and washing with balanced salt solution with oscillating; and 3, repeating freeze-thaw processes of the full-thickness cornea or the corneal stroma for 4 to 8 times and washing with balanced salt solution with oscillating to obtain the acellular cornea or the acellular corneal stroma; (3) dehydrating; and (4) sterilizing and storing. In the method, the decellularization processing time of the cornea is short; the influence on the structure and the physiological property of the cornea is small; and the processed cornea has very low immunogenicity which is similar to the property of natural cornea. The acellular cornea or the acellular corneal stroma can be applied to artificial cornea construction of tissue engineering and also can serve as a medical material applied to corneal transplantation and refraction surgery.

Description

technical field [0001] The invention belongs to the field of medical materials, and in particular relates to an acellular cornea or an acellular corneal stroma, a preparation method and application thereof. Background technique [0002] Corneal disease is the second leading cause of blindness in the world, and the number of cases is increasing at a rate of 1.5 to 2 million per year. Corneal transplantation is currently the only effective way to treat corneal blindness, but the extreme scarcity of donor corneas restricts the development of corneal transplantation. Therefore, researching and developing human corneal substitutes is the key to solving this contradiction between supply and demand. [0003] At present, it has been decades since the construction of living tissue engineered corneas in vitro that are functionally equivalent to normal corneas. Due to the unique physiological and optical properties of the cornea, it is required that the constructed tissue engineered ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61L27/36
Inventor 陈建苏戴静何克云
Owner JINAN UNIVERSITY
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