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Preparation method and application thereof of acellular conjunctiva matrix

A decellularization and conjunctival technology, which is applied in the field of preparation of acellular conjunctival matrix, can solve the problems of unfavorable cell growth, rapid degradation, easy accumulation in local areas, etc., and achieves broad application and development prospects, simple preparation method, and biocompatibility. Good results

Inactive Publication Date: 2009-12-02
SHANDONG EYE INST
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Problems solved by technology

[0003] The existing tissue engineering corneal stent materials, such as collagen and gelatin in natural polymer materials, have good biocompatibility, but have the disadvantages of poor mechanical properties and rapid degradation; artificially synthesized polymer materials such as polyglycolic acid , polylactic acid glycolic acid, etc., acidic degradation products are easy to accumulate in the local area, which is not conducive to cell growth; biologically derived materials such as "pepithelial" amniotic membrane or biological amnion, etc., degrade quickly in the body, and cannot meet the needs of tissue engineering cornea

Method used

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Examples

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preparation example Construction

[0018] Preparation method of acellular conjunctival matrix:

[0019] 1. Take fresh pig eyeballs from the slaughterhouse, wash them repeatedly with PBS, cut off the peribulbar tissue, then cut off the bulbar conjunctiva, and soak in PBS containing tobramycin 1000U / ml for 30 minutes;

[0020] 2. Cut off the Tenon's capsule of the above-mentioned bulbar conjunctiva as much as possible under the optical body condition microscope in the ultra-clean bench, rinse it with PBS, and put it in a solution with a volume fraction of 0.2% glutaraldehyde for 5 minutes for cross-linking protection;

[0021] 3. Rinse with PBS again to remove residual glutaraldehyde solution;

[0022] 4. Then decellularize the bulbar conjunctiva, act on a constant temperature shaker at 37°C with a volume fraction of 1% tritonX-100 for 24 hours, and then at 37°C, co 2 In an incubator with a concentration of 5%, act with 0.25% trypsin and 0.02% EDTA for 2 hours, rinse with PBS for 10 minutes, place in an ultra-cl...

Embodiment 1

[0041] Preparation of acellular conjunctival matrix from porcine bulbar conjunctiva:

[0042] Fresh pig eyeballs were retrieved from the slaughterhouse, rinsed repeatedly with PBS, cut off the peribulbar tissue, and then cut off the bulbar conjunctiva, soaked in PBS containing tobramycin 1000U / ml for 30 minutes; Under a stereomicroscope, cut off the Tenon's capsule of the bulbar conjunctiva as much as possible, rinse it with PBS, and put it in a solution with a volume fraction of 0.2% glutaraldehyde for 5 minutes for cross-linking protection; then rinse it with PBS to remove residual glutaraldehyde solution; then decellularize the bulbar conjunctiva, act on a constant temperature shaker at 37°C with a volume fraction of 1% tritonX-100 for 24 hours, and then at 37°C, co 2In an incubator with a concentration of 5%, act with 0.25% trypsin and 0.02% EDTA for 2 hours, rinse with PBS for 10 minutes, place in an ultra-clean table and ventilate and dry, and sterilize both sides of the...

Embodiment 2

[0044] Preparation of acellular conjunctival matrix from human bulbar conjunctiva:

[0045] Take the human bulbar conjunctiva from the eye bank and soak it in PBS containing tobramycin 1000U / ml for 30 minutes; cut off the Tenon's capsule of the above-mentioned bulbar conjunctiva as much as possible under the optical stereo microscope in the ultra-clean bench, and clean it with PBS Rinse and put in 0.2% glutaraldehyde solution for 5 minutes for cross-linking protection; then rinse with PBS to remove residual glutaraldehyde solution; then decellularize the bulbar conjunctiva and place it on a constant temperature shaker at 37°C Use a volume fraction of 1% tritonX-100 for 24 hours, then at 37°C, co 2 In an incubator with a concentration of 5%, act with 0.25% trypsin and 0.02% EDTA for 2 hours, rinse with PBS for 10 minutes, place in an ultra-clean table and ventilate and dry, and sterilize both sides of the bulbar conjunctiva with ultraviolet light for 30 minutes each. Acellular...

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Abstract

The invention relates to a preparation method and application thereof of an acellular conjunctiva matrix used as a tissue engineering corneal scaffold material. The method comprises the following steps: removing cell components in a bulbar conjunctiva first; preparing the acellular conjunctiva matrix; and taking the acellular conjunctiva matrix as a tissue engineering corneal scaffold. A rabbit corneal epithelium or an endothelial cell can form a good cell single layer on the scaffold, can construct a tissue engineering corneal epithelium or endothelium, and can successfully transplant the tissue engineering corneal epithelium or endothelium onto rabbit animal model eyes. The degradation time of the built tissue engineering cornea is longer, rabbit eyes have no obvious immune reject reaction, and the therapeutic effects on the lack of corneal limbus stem cells or the decompensation of the corneal endothelial cell function are obvious. The method and the application thereof have the advantages of simple preparation method, good biocompatibility, low antigenicity, easy growth and proliferation of seed cells, slow degradation, extensive bulbar conjunctiva sources and wide application and development prospects; and the transparency can be maintained all the time in the training process and after transplantation.

Description

technical field [0001] The invention relates to a preparation method and application of an acellular conjunctival matrix as a tissue engineering corneal scaffold material. Background technique [0002] Corneal blindness is the second most common blindness eye disease in my country. Corneal transplantation is the only effective treatment for eyesight recovery. However, the lack of corneal donor materials seriously affects the development of corneal transplantation. In recent years, with the development of tissue engineering technology, tissue engineering cornea will open up new ways for the treatment of corneal diseases. The three major elements of tissue engineering include seed cells, scaffold materials and microenvironment. The scaffold material is the place where cells live and perform functions, and at the same time guides cell growth and determines the shape of the constructed tissue. Only by choosing a good scaffold material can the seed cells grow and proliferate be...

Claims

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Application Information

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IPC IPC(8): A61L27/36A61L27/38A61F2/14A01N1/00
Inventor 史伟云赵海峰周庆军高彦高美丽杨玲玲
Owner SHANDONG EYE INST
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