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Acellular matrix with natural level of glycosaminoglycan and preparation and application thereof

A technology of acellular matrix and glycosaminoglycan, which is applied in the field of acellular matrix and its preparation, can solve the problems of biological function change, small application range, influence on cell growth, etc., achieves good biocompatibility, and improves decellularization. Efficiency, good product reproducibility

Inactive Publication Date: 2015-09-09
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages are: the natural surface structure of the acellular matrix is ​​damaged, the signal binding sites on the matrix surface are lost, and the microenvironment for cell growth is affected
However, the disadvantage of this method is that the scope of application is small and the irradiation process may cause the denaturation and inactivation of protein active substances in the matrix
The disadvantage is that the residue of the cross-linking agent will affect the growth of the cells, and it will also cause the acellular matrix to have a certain degree of hemolysis.
But there is a common problem: the bioactive components lost in the decellularization process are not replenished
Therefore, it is not difficult to find that there is a common defect that is not easy to overcome in related products in this field: the modified decellularized matrix cannot recover the biologically active components lost in a large amount during the decellularized process: glycosaminoglycans, which directly affect the Ultrastructural and extracellular matrix components of the decellularized product, resulting in altered biological function of the product

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] 1. The following processes are all aseptic operations, and the reagents used are all sterilized

[0052] I. Preparation of the carrier of the decellularized amniotic membrane cross-linked heparin of the present embodiment:

[0053] (1) Decellularization of natural biological tissues;

[0054] At room temperature, under routine aseptic operation, fresh porcine amniotic membrane (25cm 2 ) was scrubbed repeatedly with sterile normal saline to remove blood clots and serous fluid until clean, placed in a sterile square dish, and used carbonate buffer (0.05 μg / mL penicillin G and 100 μg / mL streptomycin sulfate) mol / L, pH=9.0~9.6) after immersion, place on a shaker and shake and wash for 30 minutes, repeat 4 times. Next, the amnion tissue was treated with 0.25% trypsin, 1% Triton X-100, 0.5% SDS, 0.2% glutaraldehyde and neutral protease, respectively. Specifically: ① Soak the cleaned amnion in 0.25% trypsin for 1 hour and shake it for 1 hour, wash it with PBS buffer (0.01mo...

Embodiment 2

[0071] Example 2 Preparation of acellular corneal grafted keratan sulfate carrier

[0072] 1. The following processes are all aseptic operations, and the reagents used are all sterilized

[0073] I, preparation of the carrier of the decellularized cornea grafted keratan sulfate of the present embodiment:

[0074] (1) Decellularization of natural biological tissues;

[0075]Under normal aseptic operation at room temperature, scrub the fresh rabbit cornea repeatedly with sterile saline to remove blood clots and serous fluid until it is clean, place it in a sterile square plate, and use antibiotics (100 U / mL penicillin G and 100 μg / mL sulfuric acid) Streptomycin) carbonate buffer solution (0.05mol / L, pH=9.0-9.6) soaked 5 times, 10 minutes each time. Put the cornea into sterile pure water and soak in a water bath at 37°C for 60 minutes. After soaking, put it into 10mL sterile phospholipase solution (prepared with 0.05mol / L carbonate buffer, pH=8~9; phospholipase A1=200U / mL, pho...

Embodiment 3

[0094] Example 3 Preparation of the carrier of decellularized pericardium adding hyaluronic acid

[0095] 1. The following processes are all aseptic operations, and the reagents used are all sterilized

[0096] I. Preparation of the carrier of the decellularized pericardium cross-linked hyaluronic acid of the present embodiment:

[0097] (1) Decellularization of natural biological tissues;

[0098] At room temperature, under routine aseptic operation, the fresh porcine pericardium (25cm 2 ) repeatedly scrubbed with sterile saline to remove blood clots and serous fluid until they were clean, placed them in a sterile square dish, and used carbonate buffer (0.05 μg / mL penicillin G, 100 μg / mL streptomycin sulfate) mol / L, pH=9.0~9.6) Soak 5 times, 10 minutes each time. The porcine pericardium was repeatedly frozen and thawed (frozen at -80°C, thawed at 37°C) three times, each time for 1 hour. Place 10 mL of 0.05% (w / v) trypsin-EDTA solution in a 37°C water bath for shaking (rot...

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Abstract

The invention discloses an acellular matrix with natural level of glycosaminoglycan and preparation and application thereof. The preparation method comprises the following steps: carrying out acellular disposal on natural biological tissue to obtain an acellular matrix; detecting missing amount of glycosaminoglycan in the acellular matrix; and supplementing glycosaminoglycan by means of natural ingredients in the acellular matrix to obtain the acellular matrix with natural level of glycosaminoglycan. Through the method, a lot of the glycosaminoglycan component in the acellular matrix is recovered. The acellular matrix has good biocompatibility. The natural biological structure is recovered, and the acellular matrix is beneficial to cell growth. glycosaminoglycan is supplemented for the acellular matrix, and a tissue engineering carrier is constructed. According to the invention, a biological carrier which meets cell growth requirement and mechanical strength requirement and has similar structural components as a natural extracellular matrix can be prepared rapidly. The invention is a great breakthrough of the tissue engineering construction technology. Meanwhile, principle of the invention is reliable, the technology is simple and flexible, product reproducibility is good, and industrialization is easy to realize.

Description

technical field [0001] The invention belongs to the field of tissue engineering, in particular to an acellular matrix with natural levels of glycosaminoglycans, a preparation method and application thereof. Background technique [0002] In the field of tissue engineering, the preparation of carrier scaffold materials is a key link in the construction of tissue engineering. Among them, the acellular matrix carrier has the characteristics of natural ultrafine three-dimensional structure, rich extracellular matrix components and low immunogenicity, and is widely used. A good acellular matrix carrier should have the following characteristics: ①The immune active substances are effectively removed and have good biocompatibility; ②The natural structure and components are effectively protected, and the extracellular matrix components are highly retained. As far as the preparation of acellular matrix at home and abroad is concerned, the general structure of the obtained acellular ma...

Claims

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Application Information

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IPC IPC(8): A61L27/36A61L27/20A61L27/50
Inventor 武征林熙秦子夕路程周清张建华张中夏王颖薇樊泽培严惠泽
Owner JINAN UNIVERSITY
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