Tissue engineering human corneal stroma carrier bracket and preparation method thereof

A tissue engineering and corneal stroma technology, applied in prosthesis, medical science, etc., can solve the problems of unsatisfactory biocompatibility of human corneal stromal cells, unable to meet the clinical transplantation of blind patients with corneal stromal disease, and limited experimental research, etc. Easy to attach and grow, low cost, good transparency

Active Publication Date: 2012-07-11
QINGDAO CHUNGHAO TISSUE ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the potential tumorigenicity or limited number of seed cells used, and the unsatisfactory biocompatibility between the carrier scaffold and human corneal stromal cells, the large-scale in vitro reconstruction and

Method used

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Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0021] The steps of the preparation method of the stent of the present invention are as follows:

[0022] 1) Treatment of fish skin collagen: first weigh 100-120 mg of cod skin non-enzymatic collagen freeze-dried powder, dissolve in 12-20 ml of 0.01M-0.02M hydrochloric acid, and centrifuge at 9000 rpm for 40 -50 minutes, filter the resulting supernatant with a 0.22-micron filter membrane, add 0.5-0.6 ml per well to 20 48-well plate wells, freeze at -80°C for 40-60 minutes, and then freeze-dry Machine freeze-dry for 10-12 hours.

[0023] In order to improve the toughness of the collagen composite carrier scaffold, 0.05%-0.1% of type IV collagen is added in the present invention.

[0024] In order to further improve the attachment effect of human corneal stromal cells on the collagen composite carrier scaffold, 0.05‰-0.1‰ fibronectin is added in the present invention.

[0025] 2. Preparation of crosslinking agent solution: Weigh 97.6 mg of 2-morpholineethanesulfonic acid and a...

Embodiment 1

[0028] Weigh 120 mg of cod skin non-enzymatic collagen freeze-dried powder, dissolve in 12 ml of 0.01M-0.02M hydrochloric acid, centrifuge at 9000 rpm for 50 minutes after fully dissolving, and filter the obtained supernatant with a 0.22 micron filter membrane , adding 0.6 ml per well into 20 wells of a 48-well plate, freezing at -80°C for 40 minutes, and then freeze-drying with a freeze dryer for 10 hours.

[0029] Weigh 97.6 mg of 2-morpholineethanesulfonic acid and add it to 10 ml of 40% ethanol. After fully dissolving, add 6.91 mg of N-hydroxysuccinimide. After fully dissolving, add 1-ethyl-3-(3-di 50 microliters of methylaminopropyl)-carbodiimide solution, and finally 240 mg of chondroitin sulfate was added to make a cross-linking agent solution containing 2.4% chondroitin sulfate after fully dissolving.

[0030] In the 48-well plate wells containing fish skin collagen freeze-dried powder, add a cross-linking agent solution containing 2.4% chondroitin sulfate in an amount...

Embodiment 2

[0032]Weigh 100 mg of cod skin non-enzymatic collagen freeze-dried powder, dissolve in 20 ml of 0.01M-0.02M hydrochloric acid, centrifuge at 9000 rpm for 40 minutes after fully dissolving, and filter the obtained supernatant with a 0.22 micron filter membrane , adding 0.55 ml per well into 20 wells of a 48-well plate, freezing at -80°C for 60 minutes, and then freeze-drying with a freeze dryer for 11 hours.

[0033] Weigh 97.6 mg of 2-morpholineethanesulfonic acid and add it to 10 ml of 40% ethanol. After fully dissolving, add 6.91 mg of N-hydroxysuccinimide. After fully dissolving, add 33 mM of 1-ethyl-3-(3 -60 microliters of dimethylaminopropyl)-carbodiimide solution, and finally 240 mg of chondroitin sulfate was added, and after fully dissolving, a cross-linking agent solution containing 2.4% chondroitin sulfate was prepared.

[0034] To the 48-well plate wells containing fish skin collagen freeze-dried powder, add a cross-linking agent solution containing 2.4% chondroitin ...

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PUM

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Abstract

The invention relates to a method for preparing a tissue engineering human corneal stroma carrier bracket from seawater fish collagen. The method comprises the following steps: completely dissolving non-enzymolysis codfish skin collagen freeze-dried powder into hydrochloric acid; centrifuging and removing precipitate; filling supernate into culture board pores respectively and freeze-drying; adding a 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide/N-hydroxysuccinimide crosslinking agent containing chondroitin sulfate and crosslinking; stabilizing the crosslinking effect by using an alkaline solution of Na2HPO4; cleaning by using 2M of NaCl, 1M of NaCl and distilled water sequentially; and freeze-drying to obtain the tissue engineering human corneal stroma carrier bracket. The process is scientific and reasonable; the prepared carrier bracket can be applied to mass production; heavy demand on the carrier brackets due to large-scale reconstruction of tissue engineering human corneal stromata can be met; conditions are created for sight recovery of blind people suffering from human corneal stroma diseases through clinical corneal transplantation; and the preparation cost of the carrier bracket and the application cost of the carrier bracket to in vitro reconstruction of the tissue engineering human corneal stroma and clinical treatment are low.

Description

technical field [0001] The invention belongs to the technical field of human cornea culture, and in particular relates to a tissue engineering human cornea stroma carrier bracket, that is, a tissue engineering human cornea stroma carrier bracket prepared by using seawater fish collagen. Background technique [0002] The human corneal stroma accounts for 90% of the thickness of the cornea. It is mainly composed of extracellular matrix components such as corneal stromal cells and collagen fibers. It is transparent and has a certain radius of curvature, which is conducive to the passage and bending of light. Aspects such as transparency and curvature play a crucial role. Once the corneal stroma is damaged by any trauma or inflammation, it will usually lead to corneal edema, opacity and scarring. Since there are no blood vessels in the corneal stroma, the metabolism is slow, and pathological metabolites are difficult to completely remove, even after healing. The cloudiness of t...

Claims

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Application Information

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IPC IPC(8): A61L27/24A61L27/20
Inventor 王宝泉李荣福杜玉堂樊现远郝萧
Owner QINGDAO CHUNGHAO TISSUE ENG
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