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Cartilage cell removal matrix and preparation method and application thereof

A decellularized matrix and chondrocyte technology, applied in the field of decellularized cartilage matrix and its preparation, can solve the problems of difficult growth of new cells, incomplete decellularization, and difficulty in making specific shapes

Active Publication Date: 2011-06-08
SHANGHAI TISSUE ENG LIFE SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There have been many reports on the research on the acellular matrix of cartilage tissue, but because cartilage is a relatively dense connective tissue, there are two major obstacles: incomplete decellularization and difficulty in the growth of new cells.
In order to solve the above problems, people use chemical methods to extract type II collagen, the main matrix component in cartilage, to make a gel for cartilage tissue construction, but the biomechanical strength of the gel is low, and it is difficult to make a specific shape and form a gel in the cartilage tissue. Maintain shape during construction
There are also researchers who powder cartilage tissue and then mix it with chondrocytes to build tissue, which also has the disadvantage that the shape of the tissue is difficult to control

Method used

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  • Cartilage cell removal matrix and preparation method and application thereof
  • Cartilage cell removal matrix and preparation method and application thereof
  • Cartilage cell removal matrix and preparation method and application thereof

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preparation example Construction

[0055] The preparation method of tissue engineered cartilage of the present invention comprises the steps:

[0056] In the first step, seed cells are seeded on one surface of the cartilage decellularized matrix of the present invention;

[0057] In the second step, another piece of cartilage decellularized matrix of the present invention is covered on the surface of the matrix inoculated with seed cells;

[0058] In the third step, the seed cells are seeded on another substrate covered in the second step without touching one surface of the seeded cells;

[0059] In the fourth step, the second to third steps are repeated until the superimposed cartilage decellularized matrix has 10-30 layers (preferably 15-25 layers), and the tissue-engineered cartilage graft of the present invention is obtained.

[0060] In short, the preparation method of tissue-engineered cartilage of the present invention is prepared according to the mode of "cartilage acellular matrix (sheet)-seed cells-c...

Embodiment 1

[0074] Preparation of cartilage decellularized matrix

[0075] Take fresh adult porcine auricle cartilage, cut it into a cylindrical shape with a diameter of 7 mm with a corneal trephine, cut it into circular slices with a thickness of 10-30 μm with a cryostat, put it in 1% SDS (Shanghai Shenggong) solution, and shake it on a shaker overnight. The sections were rinsed several times with distilled water, routinely stained with hematoxylin-eosin (HE) and fluorescent stained with diamidinophenylindole (DAPI) (Invitrogen, USA) to detect the degree of decellularization. Sections were freeze-dried using a freeze dryer (Virtis, USA).

[0076] result

[0077] Porcine auricular cartilage was cut into a cylindrical shape with a diameter of 7 mm using a corneal trephine ( figure 1 A, B), cryostat cut into circular slices with a thickness of 10-30 μm ( figure 1 C), after 24 hours of SDS treatment, DAPI staining found no DAPI positive staining in the 10 μm thick section, indicating tha...

Embodiment 2

[0080] Preparation of tissue engineered cartilage grafts

[0081] Auricular chondrocyte culture

[0082] The auricular cartilage tissue of newborn pigs was taken, cut into 1×1×1 mm size, digested overnight with 0.1% collagenase, filtered through a filter with a pore size of 75 μm, and then added with high-sugar DMEM (Hyclone, USA) containing 10% fetal bovine serum ( FBS; Hyclone) culture medium, placed in 5% CO 2 Cultured in a 37°C incubator, cultivated to 90% confluence and then passaged.

[0083] Construction of cartilage in vitro by sandwich method

[0084] The cultured chondrocytes were collected by digestion with 0.25% trypsin, and resuspended to different concentration groups (10, 20, 50, 100×10 6 / ml), the cartilage decellularized matrix membrane and chondrocytes were stacked layer by layer in the culture dish in the manner of "membrane-cell suspension-membrane-cell suspension" ( figure 2 ), 5 μl of cell suspension per layer, and a total of 20 layers of membranes w...

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Abstract

The invention discloses a cartilage cell removal matrix and a preparation method and application thereof. The thickness of the cartilage cell removal matrix is 1 to 30 microns, and the cartilage cell removal matrix is prepared by the following method of: (1) cutting an animal cartilage into slices with thickness of 1 to 30 microns, and (2) performing cell removal treatment on the slices obtained in the step (1). The invention also discloses a preparation method for a tissue engineered cartilage implant, which comprises the following steps of: (a) inoculating seed cells to one surface of the cartilage cell removal matrix; (b) covering the cartilage cell removal matrix of the other sheet on the surface of the matrix inoculated with the seed cells; and (c) repeating the steps (a) and (b) till the layer number of the cartilage cell removal matrix provided by the invention is 10 to 30 so as to obtain the tissue engineered cartilage implant, wherein the seed cells are selected from cartilage cells, bone marrow matrix stem cells hBMSCs or mixture thereof.

Description

technical field [0001] The invention relates to the field of tissue engineering, in particular to a cartilage decellularized matrix and its preparation method and application. Background technique [0002] Cartilage tissue defects or deformities caused by trauma, inflammation and congenital diseases are very common in clinical practice, but the self-repair ability of cartilage is low. The development of tissue engineering technology provides a good idea for repairing cartilage defects. Degradable materials are the basic elements of tissue engineered tissue components. Currently, commonly used materials for building cartilage include polylactic acid (PGA), type I collagen, and cartilage decellularized matrix. Many natural and polymer synthetic materials have been applied in the construction of cartilage tissue, but there are problems such as low biomechanical strength of materials, and immune reactions caused by degradation products. Tissue acellular matrix is ​​a natural bi...

Claims

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Application Information

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IPC IPC(8): A61L27/38A61L27/36
Inventor 张文杰龚轶一薛继鑫周广东崔磊刘伟曹谊林
Owner SHANGHAI TISSUE ENG LIFE SCI
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