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Recombinant human collagen, coding gene and application of recombinant human collagen in preparation of biodegradable collagen-based corneal substitute

A technology of human collagen and collagen-based cornea, applied in the direction of animal/human protein, application, tissue regeneration, etc., can solve the problem of corneal transplantation effectiveness that cannot take into account biocompatibility, biosafety, clinical application value, and products Difficult to market and other issues, to achieve good mechanical strength, no risk of immunogenicity, and excellent results

Pending Publication Date: 2021-12-10
山东汉肽医美生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the polymer MPDSAH and photoinitiator used in this method are stimulating reagents with poor biocompatibility, and chloroform solution is used for later storage, which is not as safe as the current terminal sterilization methods such as damp heat or radiation.
[0009] The artificial corneas disclosed in the above patents all have certain deficiencies, and cannot take into account biocompatibility, biosafety, and effectiveness after corneal transplantation. At the same time, some products are difficult to market and do not have clinical application value.

Method used

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  • Recombinant human collagen, coding gene and application of recombinant human collagen in preparation of biodegradable collagen-based corneal substitute
  • Recombinant human collagen, coding gene and application of recombinant human collagen in preparation of biodegradable collagen-based corneal substitute
  • Recombinant human collagen, coding gene and application of recombinant human collagen in preparation of biodegradable collagen-based corneal substitute

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] 1. Mix 18 g of recombinant human collagen with a molecular weight of 32 kDa and 2 g of polyvinyl alcohol 124 with a number average molecular weight of 170,000 to obtain a collagen-based blend.

[0041] 2. Dilute the collagen-based blend obtained in step 1 to 100mL with deionized water, stir evenly, and then

[0042] Add 0.02 g of carbodiimide, then place the resulting mixture in a mold, and conduct a crosslinking reaction at 25° C. for 2 hours.

[0043] 3. Put the sample after the cross-linking reaction in step 2 into a 1kDa cut-off dialysis bag for cross-linking agent removal treatment, and replace the ultra-pure water in the external environment every 6 hours, a total of 4 times; the residual cross-linking agent will be removed in a clean environment Samples were placed in phosphate sterile preservation solution and packaged, treated at -10°C for 8 hours, 10kGy Co 60 Sterilized by irradiation to obtain biodegradable collagen-based corneal substitutes.

[0044] 4. Ad...

Embodiment 2

[0047] 1. Mix 8 g of recombinant human collagen with a molecular weight of 32 kDa and 8 g of polyvinyl alcohol 124 with a number average molecular weight of 120,000 to obtain a collagen-based blend.

[0048] 2. Dilute the collagen-based blend obtained in step 1 to 100mL with deionized water, stir well, then add 0.032g of transglutaminase, and then place the resulting mixture in a mold for cross-linking reaction at 4°C 48h; the obtained product was washed with PBS for 30min, then added to 100mL of deionized water, stirred evenly, added 0.016g of glutaraldehyde, and cross-linked at 25°C for 4h.

[0049] 3. Ultrasonic cleaning of the sample after the cross-linking reaction in step 2 to remove the cross-linking agent, and then place the sample in a clean environment in a phosphate sterile preservation solution and package it, and treat it at -20°C for 6 hours. 15kGyCo 60 Sterilized by irradiation to obtain biodegradable collagen-based corneal substitutes.

[0050] 4. Add 1mL of ...

Embodiment 3

[0053] 1. Mix 8 g of recombinant human collagen freeze-dried powder with a molecular weight of 32 kDa, 1 g of polylactic acid with a number average molecular weight of 40,000, and 1 g of polyglycolic acid with a number average molecular weight of 20,000 to obtain a collagen-based blend.

[0054] 2. The above-mentioned collagen-based blend was formed into a scaffold by electrospinning technology to obtain a nanoscale three-dimensional mesh scaffold. The scaffold material was cut to the required size and immersed in 100mL ethanol solution containing 0.3g carbodiimide , cross-linking reaction at 25°C for 10h; the obtained product was rinsed with PBS for 30min, then immersed in 100mL aqueous solution containing 0.2g transglutaminase, and cross-linking reaction at 4°C for 16h.

[0055] 3. The corneal stent after the cross-linking reaction in step 2 was repeatedly washed with PBS to remove the remaining cross-linking agent. After freeze-drying, it was packaged in a medical aluminum f...

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Abstract

The invention discloses a recombinant human collagen, a coding gene and an application of the recombinant human collagen in preparation of a biodegradable collagen-based corneal substitute, and the recombinant human collagen and the coding gene are obtained by treating the recombinant human collagen and a high-molecular polymer in a water phase in a composite cross-linking manner and curing and molding in a mold, or after the recombinant human collagen and the high-molecular polymer are subjected to electrostatic spinning to form a nanofiber membrane, the nanofiber membrane is further subjected to composite crosslinking and drying to obtain the three-dimensional corneal scaffold, and platelet-rich plasma is added for use after rehydration swelling in clinical application. The cornea substitute is free of virus hidden danger, free of immunogenicity, controllable in degradability and good in refraction and mechanical strength, and has a remarkable induction effect on regeneration of corneal epithelial cells, corneal bodies and corneal nerves after being added with platelet-rich plasma (APRP).

Description

technical field [0001] The invention belongs to the technical field of tissue engineering corneal graft materials, in particular, the invention relates to a recombinant human collagen, a coding gene and its application in the preparation of a biodegradable collagen-based corneal substitute. Background technique [0002] The blindness of more than 10 million people in the world is caused by corneal disease, which is the second leading cause of blindness after cataract. Penetrating keratoplasty is the main treatment for corneal diseases, but it has a poor prognosis for severe dry eye, Stevens-Johnson syndrome, severe chemical burns, etc., and faces problems such as graft rejection and insufficient donors. The application of artificial corneas and the research on tissue engineering corneas have brought hope to blind patients. At present, the commonly used corneal construction materials are mainly bio-scaffold materials and polymer materials. [0003] Biomaterial scaffolds ofte...

Claims

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Application Information

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IPC IPC(8): C07K14/78C12N15/12A61L27/24A61L27/16A61L27/18A61L27/36A61L27/50A61L27/58D06M13/432D06M101/14
CPCC07K14/78A61L27/24A61L27/16A61L27/18A61L27/3616A61L27/3641A61L27/50A61L27/58D06M13/432A61L2430/16A61L2400/12D06M2101/14C08L29/04C08L67/04
Inventor 殷世清赵学彩张凤龙蔡永刚魏静孙晖李倩张甲庆李鑫
Owner 山东汉肽医美生物科技有限公司
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