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Preparation method of multiple decellularized material of porcine small intestinal submucosa modified by nucleus pulposus cells

A technology of nucleus pulposus cells and submucosa, which is applied in the field of lumbar intervertebral disc tissue repair and regeneration, and achieves the effects of good injectability, good extracellular microenvironment, and enhanced repair effect

Active Publication Date: 2020-11-13
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no one has reported the use of acellular SIS to repair intervertebral disc degeneration

Method used

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  • Preparation method of multiple decellularized material of porcine small intestinal submucosa modified by nucleus pulposus cells
  • Preparation method of multiple decellularized material of porcine small intestinal submucosa modified by nucleus pulposus cells
  • Preparation method of multiple decellularized material of porcine small intestinal submucosa modified by nucleus pulposus cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Preparation and research of porcine SIS multiple decellularized material modified by nucleus pulposus

[0042] (1) The upper segment of porcine jejunum was rinsed with sterile normal saline for 3 times for 20 minutes each time to remove the mucous membrane and muscular layer, and then rinsed with sterile normal saline for 3 times for 20 minutes each time; the sample size was usually 5cm x 5cm.

[0043] (2) Cool the SIS completely in liquid nitrogen, and then place it in a water bath at 37°C for half an hour to restore the temperature; repeat the above process 10 times to facilitate subsequent reagent diffusion;

[0044] (3) In 1000ml of physiological saline buffer containing protease inhibitors (concentration is 1%, protease inhibitor content is 10KIU / ml), shake at 120rpm on a shaker at a constant temperature of 45°C for 24 hours, and rinse with normal saline for 5 hours;

[0045] (4) In 1000ml of organic solvent solution (chloroform and methanol solution wit...

Embodiment 2

[0056] Example 2 Preparation and research of porcine SIS multiple decellularized material modified by nucleus pulposus

[0057] Get the pig SIS tissue, step (3) in 1000ml of physiological saline buffer containing protease inhibitor (concentration is 3%, protease inhibitor content is 10KIU / ml), constant temperature 45 DEG C shaker 120rpm shakes 8 hours; Step (12 ) In 1000ml of SDS-containing PBS buffer solution (SDS concentration is 3%, mixed with 10mmol of Tris), add 100ml of penicillin and streptomycin mixed antibacterial liquid, shake at a constant temperature of 45°C at 120rpm for 15 hours; the rest refer to Example 1 The method was carried out to obtain multiple decellularized injectable materials of porcine nucleus pulposus-modified porcine SIS.

Embodiment 3

[0058] Example 3 Preparation and research of porcine SIS multiple decellularized material modified by nucleus pulposus

[0059] The porcine SIS tissue was taken, and the rest were carried out with reference to the method of Example 1 to obtain the multiple decellularized injectable material of porcine nucleus pulposus modified.

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Abstract

The invention discloses a preparation method of multiple acellular materials modified with nucleus pulposus cells for porcine small intestinal submucosa (SIS). The method specially comprises the following steps: stripping the porcine SIS tissues; carrying out treatment on the stripped porcine SIS tissues through a normal saline buffer solution containing a protease inhibitor, an organic solvent solution, a PBS (Phosphate Buffered Saline) buffer solution containing Triton X, a PBS buffer solution containing SDS (Sodium Dodecyl Sulfate) and a PBS buffer solution containing DNA enzyme to obtain acellular SIS materials; performing ball milling and pulverization on the acellular SIS materials to obtain micro-particles with diameter of 200mu m; carrying out co-culture on the micro-particles andseparated nucleus pulposus cells and then carrying out decellularization treatment again so as to obtain injectable multiple acellular nucleus pulposus repairing materials. According to the preparation method of the multiple acellular materials modified with the nucleus pulposus cells for the porcine SIS disclosed by the invention, the completeness of original ECM (Extra Cellular Matrix) can be preserved while allogeneic cells or heterologous cells with immunoreactivity are removed at the same time, and the preparation method of the multiple acellular materials modified with the nucleus pulposus cells for the porcine SIS has an good extracellular micro-environment, bio-factors, biomechanical properties and the like, and can simulate a growth environment of nucleus pulposus cells in normalphysiological conditions to a maximum limit.

Description

technical field [0001] The invention belongs to the technical field of lumbar intervertebral disc tissue repair and regeneration, and in particular relates to a natural tissue-derived and natural cell-modified multiple decellularized nucleus pulposus repair material and a preparation method thereof. Background technique [0002] The incidence of low back pain in the population is as high as 60%, and a considerable part of it is closely related to lumbar disc degeneration. Although lumbar disc degeneration can be treated clinically through various surgical or non-surgical methods, methods such as pain control and spinal fusion can only relieve symptoms, but cannot rebuild the spinal structure. For this reason, artificial intervertebral disc repair materials are the direction of current research, but due to biocompatibility, difficulty in regeneration of nucleus pulposus cells, etc., there is still no suitable material that can really be used for the treatment of intervertebra...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61L27/36A61L27/50
CPCA61L27/3629A61L27/3691A61L27/3695A61L27/50A61L2400/06A61L2430/38
Inventor 单治林贤丰王晟毓范顺武赵凤东
Owner ZHEJIANG UNIV
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