Articular cartilage graft and preparation method thereof

Abstract

An articular cartilage graft and a preparation method thereof are provided. The prepared articular cartilage graft is composed of a superficial layer, a middle layer and a deep layer from outside to inside; the thickness, shape and size of each layer are all matched with a cartilage injury part, and thus preoperative shaping is not required; after grafting, the articular cartilage graft has layer distribution corresponding to distribution of each layer of surrounding normal cartilages, is conducive to intercellular signal transmission, transduction and regulation, and can be better integrated with surrounding normal cartilage tissues; the articular cartilage graft has structure characteristics consistent with those of the natural cartilages, the collagen type II content is gradually decreased from the superficial layer to the deep layer, the GAG content is increased gradually from the superficial layer to the deep layer, and compression resistance and wear resistance are good; and chondrocytes in the cartilage graft are wrapped with an extracellular matrix, have low immunogenicity, allow generation of immunologic rejection to be avoided after grafting, can survive for a long term and exert functions, and improve cartilage repair long-term curative effects.

Description

technical field

[0001] The invention belongs to the technical field of tissue engineering medical biomaterials, and in particular relates to an articular cartilage graft and a preparation method thereof. Background technique

[0002] Articular cartilage is hyaline cartilage composed of chondrocytes, cartilage fibers, and cartilage matrix. Cartilage tissue has the characteristics of heterogeneous layer distribution, which is divided into superficial layer (10-20%), middle layer (40-60%) and deep layer (20-30%) from top to bottom. In recent years, studies have found that each region of cartilage has a different structure, different protein components, and performs different functions. The gene expression and growth rate of chondrocytes obtained from different regions are different. The density of chondrocytes in the superficial, middle and deep layers decreases in turn, among which the chondrocytes in the superficial layer are closely arranged in the horizontal direction and ...

Examples

Embodiment 1

[0030] Step 1. Obtain chondrocytes: cut the rabbit superficial cartilage tissue into 0.5~3mm 3 Size, first digest with 10 times volume of 0.1% (w / v) hyaluronidase solution for 30 minutes, shake once every 15 minutes, rinse with PBS twice after digestion; then add 10 times volume of 0.2% (w / v) ) Digestion with type II collagenase solution for 4 hours, stop the digestion with PBS and fully pipette the cells, filter with a 200-mesh sieve, and wash twice with PBS to isolate the primary superficial chondrocytes at a cell density of 1×10 5 Pcs / cm 2 Inoculate in culture flask, add culture solution A, 37℃, 5% CO 2 After culturing in the incubator for 3 days, when the superficial chondrocytes reach 80% confluence, they are subcultured. Add 10 times the volume of 0.25% (w / v) trypsin solution to digest for 3 minutes, stop with PBS and wash twice, according to the cell density of 1×10 4 Pcs / cm 2 Inoculate, add culture medium A, and subculture once every 3 days;

[0031] Cut the rabbit middle c...

Embodiment 2

[0043] Step 1. Obtain chondrocytes: cut the superficial layer of porcine cartilage into 0.5~3mm 3 Size, first digest with 10 times volume of 0.15% (w / v) hyaluronidase solution for 45 minutes, shake once every 15 minutes, rinse with PBS twice after digestion; then add 10 times volume of 0.25% (w / v) v) Digestion with type II collagenase solution for 8 hours, stop the digestion with PBS and fully pipette, filter with 200 mesh screen and wash twice with PBS to isolate primary superficial chondrocytes at a cell density of 5×10 5 Pcs / cm 2 Inoculate in culture flask, add culture solution A, 37℃, 5% CO 2 After 7 days of culture in the incubator, when chondrocytes reach more than 90% confluence, pass them down, digest with 10 times the volume of 0.5% (w / v) trypsin solution for 8 minutes, stop with PBS and wash twice, according to cell density 1×10 5 Pcs / cm 2 Inoculate, add culture medium A, passage once every 7 days, and change the medium every 3 days;

[0044] Cut the porcine middle cartil...

technical field

[0001] The invention belongs to the technical field of tissue engineering medical biomaterials, and in particular relates to an articular cartilage graft and a preparation method thereof. Background technique

[0002] Articular cartilage is hyaline cartilage composed of chondrocytes, cartilage fibers, and cartilage matrix. Cartilage tissue has the characteristics of heterogeneous layer distribution, which is divided into superficial layer (10-20%), middle layer (40-60%) and deep layer (20-30%) from top to bottom. In recent years, studies have found that each region of cartilage has a different structure, different protein components, and performs different functions. The gene expression and growth rate of chondrocytes obtained from different regions are different. The density of chondrocytes in the superficial, middle and deep layers decreases in turn, among which the chondrocytes in the superficial layer are closely arranged in the horizontal direction and ...

Examples

Embodiment 1

[0030] Step 1. Obtain chondrocytes: cut the rabbit superficial cartilage tissue into 0.5~3mm 3 Size, first digest with 10 times volume of 0.1% (w / v) hyaluronidase solution for 30 minutes, shake once every 15 minutes, rinse with PBS twice after digestion; then add 10 times volume of 0.2% (w / v) ) Digestion with type II collagenase solution for 4 hours, stop the digestion with PBS and fully pipette the cells, filter with a 200-mesh sieve, and wash twice with PBS to isolate the primary superficial chondrocytes at a cell density of 1×10 5 Pcs / cm 2 Inoculate in culture flask, add culture solution A, 37℃, 5% CO 2 After culturing in the incubator for 3 days, when the superficial chondrocytes reach 80% confluence, they are subcultured. Add 10 times the volume of 0.25% (w / v) trypsin solution to digest for 3 minutes, stop with PBS and wash twice, according to the cell density of 1×10 4 Pcs / cm 2 Inoculate, add culture medium A, and subculture once every 3 days;

[0031] Cut the rabbit middle c...

Embodiment 2

[0043] Step 1. Obtain chondrocytes: cut the superficial layer of porcine cartilage into 0.5~3mm 3 Size, first digest with 10 times volume of 0.15% (w / v) hyaluronidase solution for 45 minutes, shake once every 15 minutes, rinse with PBS twice after digestion; then add 10 times volume of 0.25% (w / v) v) Digestion with type II collagenase solution for 8 hours, stop the digestion with PBS and fully pipette, filter with 200 mesh screen and wash twice with PBS to isolate primary superficial chondrocytes at a cell density of 5×10 5 Pcs / cm 2 Inoculate in culture flask, add culture solution A, 37℃, 5% CO 2 After 7 days of culture in the incubator, when chondrocytes reach more than 90% confluence, pass them down, digest with 10 times the volume of 0.5% (w / v) trypsin solution for 8 minutes, stop with PBS and wash twice, according to cell density 1×10 5 Pcs / cm 2 Inoculate, add culture medium A, passage once every 7 days, and change the medium every 3 days;

[0044] Cut the porcine middle cartil...

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