Injectable mesenchymal stem cell extracellular matrix/agarose composite hydrogel as well as preparation method and application thereof

A technology of bone marrow mesenchyme and composite hydrogel, applied in the direction of bone/connective tissue cells, animal cells, vertebrate cells, etc., can solve problems such as difficult to effectively promote cell adhesion, proliferation and differentiation, failure to provide, etc. Achieve the effects of increasing cell adhesion and plasticity, facilitating repair, and sufficient cell sources

Inactive Publication Date: 2015-01-28
南京医科大学附属南京医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the use of agarose hydrogel simulates the water environment of the natural extracellular matrix, it fails to provide the functional, structural

Method used

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  • Injectable mesenchymal stem cell extracellular matrix/agarose composite hydrogel as well as preparation method and application thereof
  • Injectable mesenchymal stem cell extracellular matrix/agarose composite hydrogel as well as preparation method and application thereof
  • Injectable mesenchymal stem cell extracellular matrix/agarose composite hydrogel as well as preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0034] (1) Schematic diagram of the preparation process of injectable bone marrow mesenchymal stem extracellular matrix / agarose composite hydrogel, see figure 1 , the specific method is as follows:

[0035] (1) Isolation and cultivation of rabbit bone marrow mesenchymal stem cells by density gradient centrifugation: extract bone marrow from the posterior superior iliac spine of 4-month-old New Zealand white rabbits, add an equal volume of normal saline, mix well, and slowly adhere to the wall and inject with Centrifuge the same volume of bone marrow lymphocyte separation liquid in a centrifuge tube at 1200 rpm for 10 minutes. After centrifugation, the mononuclear cell layer was collected and counted at 1 × 10 8 The cell density was seeded in a petri dish with a diameter of 150 mm (25 ml of DMEM culture solution containing 10% fetal bovine serum was added to each petri dish in advance), and placed at 37°C, 5% CO 2 Cultured in a saturated humidity incubator. After the cells w...

Embodiment 2

[0041] Take the rabbit bone marrow mesenchymal stem cell extracellular matrix suspension collected in Example 1, while preparing the bone marrow mesenchymal stem extracellular matrix / agarose composite hydrogel, wrap the chondrocytes in the composite hydrogel, General observations at different culture time points, the results are shown in Figure 5 ; and observe the safranin O staining of the cultured tissues at different culture time points, the results are shown in Figure 6 .

[0042] (1) Digest the chondrocytes that have passed to P2 or P3 with 0.25% trypsin, count and control the cell density to be 5×10 6 pieces / ml.

[0043] (2) Mix the digested chondrocyte suspension with the rabbit bone marrow mesenchymal stem cell extramatrix suspension, then mix with 4% agarose, shake gently to make it fully mixed, and obtain the bone marrow mesenchymal stem cells wrapped in chondrocytes Stem extracellular matrix / agarose composite hydrogel suspension. Next, the composite hydrogel s...

Embodiment 3

[0063] Injectable autologous bone marrow mesenchymal stem cell extramatrix / agarose composite hydrogel for repairing cartilage defects Figure 7 , the specific method is as follows:

[0064] (1) Prepare a cartilage defect with a diameter of 3mm at the femoral trochlear of the rabbit with a biological puncher ( Figure 7 A);

[0065] (2) Prepare several microfracture holes in the cartilage defect with injection needles ( Figure 7 B);

[0066] (3) Take the bone marrow mesenchymal stem cell extracellular matrix suspension collected in Example 1, mix it with 4% agarose in an equal volume, shake it gently to make it fully mixed, and obtain the rabbit bone marrow mesenchymal stem extracellular matrix / Agarose composite hydrogel, then use a syringe to drop the obtained rabbit bone marrow mesenchymal stem cell extracellular matrix / agarose composite hydrogel to the cartilage defect, and set the agarose hydrogel repair group at the same time (for the preparation method of agarose hyd...

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Abstract

The invention discloses injectable mesenchymal stem cell extracellular matrix/agarose composite hydrogel as well as a preparation method and an application thereof. The preparation method comprises the following steps: culturing mesenchymal stem cells of a rabbit in vitro with monolayer high density, collecting a secreted extracellular matrix of the mesenchymal stem cells, and acquiring an extracellular matrix suspension by utilizing freeze drying, grinding under liquid nitrogen and an enzymatic digestion technology; and then fully and uniformly mixing the extracellular matrix suspension and agarose with same volume to prepare the mesenchymal stem cell extracellular matrix/agarose composite hydrogel material. The injectable mesenchymal stem cell extracellular matrix/agarose composite hydrogel can be injected into cartilage defect parts in arbitrary shapes by virtue of a simple injection mode and has good adhesion integration capability with peripheral host tissues,is sufficient in cell source, avoids potential risks caused by xenogenic resource materials, and is convenient to repair cartilage defects in arbitrary shapes to reach purpose of good integration with the peripheral host tissues.

Description

technical field [0001] The invention belongs to the field of biological materials, and relates to an injectable bone marrow mesenchymal stem cell extracellular matrix / agarose composite hydrogel, a preparation method and application thereof. Background technique [0002] Articular cartilage belongs to hyaline cartilage without innervation or blood vessel distribution, so the self-repair ability of damaged articular cartilage is limited. After the articular cartilage is damaged, the absorption and cushioning effect of the stress is reduced, and the damage will be progressively aggravated. At the same time, the damaged articular cartilage has the risk of developing severe osteoarthritis. At present, the repair methods of articular cartilage defects mainly include: joint washing and debridement, microfracture bone marrow stimulation, osteochondral transplantation and autologous chondrocyte transplantation. The above-mentioned technologies have their own advantages and disadvant...

Claims

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Application Information

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IPC IPC(8): A61L27/36A61L27/22A61L27/20A61L27/52C12N5/0775
Inventor 王黎明魏波徐燕
Owner 南京医科大学附属南京医院
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