Acellular cartilage matrix and preparation method thereof

Abstract

The invention relates to the technical field of articular cartilages, in particular toarticular cartilage matrix and a preparation method thereof. Cartilage tissues are sequentially subjected to hypotonic treatment, trypsin treatment, detergent treatment and nuclease treatment to obtain the cartilage matrix. The prepared acellular cartilage matrix can maintain the three-dimensional structure of chondrocyte matrix itself to the maximum extent, have good mechanical properties, do not require an external crosslinking agent for physical or chemical crosslinking, have low immunogenicity and low toxicity, have the simple preparation method and low cost, and be suitable for industrial production.

Description

technical field

[0001] The invention relates to the technical field of articular cartilage, in particular to a decellularized cartilage matrix and a preparation method thereof. Background technique

[0002] Articular cartilage is an important structure of human joints. The main function of articular cartilage is to protect the articular surface of bones from wear and tear. Because there are no blood vessels, lymph and cartilage distribution on the articular cartilage, and the chondrocytes are highly differentiated, the self-repair ability of the articular cartilage is extremely limited. Once the articular cartilage is destroyed, the pathological changes are often irreversible, regeneration is very difficult, and cartilage damage is severe It affects people's normal walking and daily activities, and long-term cartilage damage is also one of the main causes of physical disabilities.

[0003] Decellularization technology is a method of removing immunogenic tissue cells and ret...

Examples

Embodiment 1

[0045] The preparation method of the decellularized cartilage matrix in this embodiment comprises the following steps:

[0046] (1) Pre-cleaning treatment: Take 800g of fresh cartilage tissue and add it to normal saline, soak it, then take out the cartilage tissue and add it to ultrapure water, ultrasonically clean it with an ultrasonic cleaner, and then rinse it continuously with ultrapure water.

[0047] (2) Repeated freezing and thawing treatment: the cartilage tissue was first frozen at -80°C for 3 hours, then thawed at room temperature for 4 hours, and repeated freezing and thawing 4 times.

[0048] (3) Hypotonic treatment: add cartilage tissue to 10L Tris-Hcl solution that has been sterilized, and incubate at room temperature for 20 hours. Wherein, the pH of the Tris-Hcl solution is 8, and the concentration is 10 mM.

[0049] (4) Trypsin treatment: put the cartilage tissue into a trypsin solution, and heat in a water bath at 37° C. for 8 hours. Wherein, the trypsin sol...

Embodiment 2

[0059] The preparation method of the decellularized cartilage matrix in this embodiment comprises the following steps:

[0060] (1) Pre-cleaning treatment: Take 1 kg of fresh cartilage tissue and add it to normal saline, soak it, then take out the cartilage tissue and add it to ultrapure water, ultrasonically clean it with an ultrasonic cleaner, and then rinse it continuously with ultrapure water.

[0061] (2) Repeated freezing and thawing treatment: the cartilage tissue was first frozen at -80°C for 2 hours, then thawed at room temperature for 6 hours, and repeated freezing and thawing 6 times.

[0062] (3) Hypotonic treatment: add cartilage tissue into sterilized Tris-Hcl solution, and incubate at room temperature for 15 hours. Wherein, the pH of the Tris-Hcl solution is 8, the volume is 10 L, and the concentration is 10 mM.

[0063] (4) Trypsin treatment: the cartilage tissue was washed with ultrapure water, put into trypsin solution, and heated in a water bath at 37° C. f...

Embodiment 3

[0073] The preparation method of the decellularized cartilage matrix in this embodiment comprises the following steps:

[0074] (1) Pre-cleaning treatment: Take 500g of fresh cartilage tissue and add it to normal saline, soak it, then take out the cartilage tissue and add it to ultrapure water, ultrasonically clean it with an ultrasonic cleaner, and then rinse it continuously with ultrapure water.

[0075] (2) Repeated freezing and thawing treatment: the cartilage tissue was first frozen at -70°C for 4 hours, then thawed at 30°C for 1 hour, and repeated freezing and thawing twice.

[0076] (3) Hypotonic treatment: the cartilage tissue was added to a sterilized 8 mM Tris-Hcl solution, and incubated at room temperature for 30 h. Wherein, the pH of the Tris-Hcl solution is 8, and the volume is 10 L.

[0077] (4) Trypsin treatment: the cartilage tissue was washed with ultrapure water, put into trypsin solution, and heated in a water bath at 37° C. for 24 hours. Wherein, the tryp...

technical field

[0001] The invention relates to the technical field of articular cartilage, in particular to a decellularized cartilage matrix and a preparation method thereof. Background technique

[0002] Articular cartilage is an important structure of human joints. The main function of articular cartilage is to protect the articular surface of bones from wear and tear. Because there are no blood vessels, lymph and cartilage distribution on the articular cartilage, and the chondrocytes are highly differentiated, the self-repair ability of the articular cartilage is extremely limited. Once the articular cartilage is destroyed, the pathological changes are often irreversible, regeneration is very difficult, and cartilage damage is severe It affects people's normal walking and daily activities, and long-term cartilage damage is also one of the main causes of physical disabilities.

[0003] Decellularization technology is a method of removing immunogenic tissue cells and ret...

Examples

Embodiment 1

[0045] The preparation method of the decellularized cartilage matrix in this embodiment comprises the following steps:

[0046] (1) Pre-cleaning treatment: Take 800g of fresh cartilage tissue and add it to normal saline, soak it, then take out the cartilage tissue and add it to ultrapure water, ultrasonically clean it with an ultrasonic cleaner, and then rinse it continuously with ultrapure water.

[0047] (2) Repeated freezing and thawing treatment: the cartilage tissue was first frozen at -80°C for 3 hours, then thawed at room temperature for 4 hours, and repeated freezing and thawing 4 times.

[0048] (3) Hypotonic treatment: add cartilage tissue to 10L Tris-Hcl solution that has been sterilized, and incubate at room temperature for 20 hours. Wherein, the pH of the Tris-Hcl solution is 8, and the concentration is 10 mM.

[0049] (4) Trypsin treatment: put the cartilage tissue into a trypsin solution, and heat in a water bath at 37° C. for 8 hours. Wherein, the trypsin sol...

Embodiment 2

[0059] The preparation method of the decellularized cartilage matrix in this embodiment comprises the following steps:

[0060] (1) Pre-cleaning treatment: Take 1 kg of fresh cartilage tissue and add it to normal saline, soak it, then take out the cartilage tissue and add it to ultrapure water, ultrasonically clean it with an ultrasonic cleaner, and then rinse it continuously with ultrapure water.

[0061] (2) Repeated freezing and thawing treatment: the cartilage tissue was first frozen at -80°C for 2 hours, then thawed at room temperature for 6 hours, and repeated freezing and thawing 6 times.

[0062] (3) Hypotonic treatment: add cartilage tissue into sterilized Tris-Hcl solution, and incubate at room temperature for 15 hours. Wherein, the pH of the Tris-Hcl solution is 8, the volume is 10 L, and the concentration is 10 mM.

[0063] (4) Trypsin treatment: the cartilage tissue was washed with ultrapure water, put into trypsin solution, and heated in a water bath at 37° C. f...

Embodiment 3

[0073] The preparation method of the decellularized cartilage matrix in this embodiment comprises the following steps:

[0074] (1) Pre-cleaning treatment: Take 500g of fresh cartilage tissue and add it to normal saline, soak it, then take out the cartilage tissue and add it to ultrapure water, ultrasonically clean it with an ultrasonic cleaner, and then rinse it continuously with ultrapure water.

[0075] (2) Repeated freezing and thawing treatment: the cartilage tissue was first frozen at -70°C for 4 hours, then thawed at 30°C for 1 hour, and repeated freezing and thawing twice.

[0076] (3) Hypotonic treatment: the cartilage tissue was added to a sterilized 8 mM Tris-Hcl solution, and incubated at room temperature for 30 h. Wherein, the pH of the Tris-Hcl solution is 8, and the volume is 10 L.

[0077] (4) Trypsin treatment: the cartilage tissue was washed with ultrapure water, put into trypsin solution, and heated in a water bath at 37° C. for 24 hours. Wherein, the tryp...

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