Preparing method and application of acellular amniotic membrane used for repairing skin wound difficult to heal

Abstract

The invention relates to a preparing method and application of an amniotic membrane, in particular to a preparing method and application of an acellular amniotic membrane used for repairing skin wound difficult to heal. The preparing method specifically comprises the steps of isolating, cleaning and sterilizing an amniotic membrane tissue; arranging and drying the amniotic membrane tissue on a nitrocellulose membrane to manufacture an amniotic membrane paster; vibrating and digesting the amniotic membrane paster in mixed digestive fluid of 0.25%-0.5% of pancreatin and 0.2-0.5g / L EDTA.4Na for 10-30 minutes at a temperature of 37 DEG C; rinsing the amniotic membrane paster in TE buffer solution; vibrating overnight to fully remove amniotic membrane cells in TE-TritonX-100 solution; washing the amniotic membrane paster for three times in TE solution; vibrating and degrading the amniotic membrane paster for 2-4 hours in nucleic acid degrading solution at a temperature of 37 DEG C; washing the amniotic membrane paster for three times in the TE solution. The prepared acellular amniotic membrane paster can be stored for a long time after being refrigerated, dried, packed and sterilized by cobalt 60 irradiation and is a tissue engineering material which can be taken and used when needed. The acellular amniotic membrane paster can be easily separated from the nitrocellulose membrane to be used for repairing the skin wound after rewatered.

Description

technical field [0001] The invention relates to a preparation method and application of amniotic membrane. Background technique [0002] Amnion tissue discarded during the perinatal period is one of the important sources of seed cells for regenerative medicine. It has a wide range of sources, is easy to obtain, and has no ethical and moral disputes. It has become a research hotspot in recent years. Amnion, as an immune-exempt biological graft, has biological properties such as anti-inflammation, anti-adhesion, pain relief, and promotion of epithelialization. It has been applied to burn patients and other surgical operations as early as 1910 to promote epidermalization, Reduce pain and prevent infection. In recent years, with the further understanding of amniotic membrane and the development of amniotic membrane preservation and preparation technology, amniotic membrane has once again become a research and application hotspot, and is widely used in corneal reconstruction, pl...

AI Technical Summary

Problems solved by technology

However, the existing amniotic membrane decellularization method still cannot guarantee the integrity of the amniotic matrix on the basis of complete removal of cells, especially it will cause serious damage to the basement membrane, affecting the adhesion and migration of seed cells or endogenous cells in vivo

Patent Publication No. CN1757717A (preparation method of dry active amniotic membrane) uses trypsin to treat for a long time in combination with cell scraper or cotton swab to remove epithelial cells, but long-term trypsin digestion will lead to changes or even degradation of amnion collagen structure, while physical scraping Injury to basement membrane components that severely disrupt amnion epithelial cell adhesion

Patent Publication No. CN102327640A (a preparation method of decellularized amnion) uses SDS for cell lysis to promote the detachment of amnion epithelial cells, but low concentration of SDS will limit the removal efficiency of amnion cells, but high concentration of SDS will cause damage to the amnion matrix

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