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Synovial mesenchymal stem cell and PRP combined preparation for repairing articular cartilage injury as well as preparation method and application thereof

A technique for stromal stem cells and cartilage damage, which is applied in the field of synovial mesenchymal stem cells combined with PRP preparations, synovial mesenchymal stem cells combined with PRP preparations and their preparation fields, which can solve the disorder of joint alignment, the long-term effect is not obvious, and there is no obvious long-term effect. It can repair articular cartilage damage, high chondrocyte proliferation, and no immunogenicity.

Pending Publication Date: 2021-02-02
国大生命科学产业集团(深圳)有限公司
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  • Summary
  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

Drug treatment includes anti-inflammatory and analgesic drugs and intracavitary local injection of corticosteroids. Non-steroidal anti-inflammatory drugs cannot change the process of joint degenerative diseases and can only relieve pain, but they will bring many side effects, such as common gastrointestinal tract bleeding; intra-articular injection of viscosupplements is helpful for the treatment of symptomatic knee osteoarthritis, such as sodium hyaluronate. There is no obvious effect on bone contact and obvious joint force line disorder;
[0005] 2. Surgical treatment: There are many surgical methods for the treatment of cartilage defects. The success rate depends on the location, size, shape and depth of the defect, as well as the age and activity level of the patient. Surgical therapy has technical limitations and is currently only applicable for certain patients
Joint lavage and debridement refer to flushing the diseased joints to achieve the purpose of treatment. Through a large amount of saline flushing, debris and inflammatory mediators can be removed, which can delay the progress of joint inflammation, and the long-term effect is not obvious; bone marrow stimulation is Refers to the use of various methods to penetrate the subchondral bone in the cartilage injury area, stimulate the subchondral bone to bleed, and form a fibrin clot to fill the defect area. This heterogeneous repair tissue has poor mechanical properties and will degenerate over time, especially It is an uncorrected alignment deformity; autologous chondrocyte transplantation is to inject autologous chondrocytes into the joint cavity of damaged cartilage, and its repair effect is good, but the operation is difficult and requires additional surgical injuries
[0006] With the development of stem cell technology, there are more and more clinical studies of stem cells in the treatment of various diseases, because they have self-renewal and can differentiate into various types of cells and can secrete a variety of stem cell factors to activate cell proliferation and immune regulation in the body The function of inhibiting inflammation has a wide range of application prospects, so it is also used as the main research object for the treatment of articular cartilage injury. However, direct injection of mesenchymal stem cells to treat articular cartilage injury has the disadvantages of low cell survival rate and difficult cell proliferation.

Method used

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  • Synovial mesenchymal stem cell and PRP combined preparation for repairing articular cartilage injury as well as preparation method and application thereof
  • Synovial mesenchymal stem cell and PRP combined preparation for repairing articular cartilage injury as well as preparation method and application thereof
  • Synovial mesenchymal stem cell and PRP combined preparation for repairing articular cartilage injury as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Isolation and cultivation of synovial mesenchymal stem cells:

[0053] Under aseptic conditions, the synovial tissue was removed from the suprapatellar bursa of the knee joint with forceps, and the collected specimen was immersed in sterile saline at 4°C;

[0054] Move the specimen into the aseptic operating table, wash it with normal saline for 3 times, cut off the fat and some connective tissue, separate the smooth and bright synovium tissue, and cut the synovium into 1.0-2.0 mm with sterile ophthalmic scissors 3 Fragmentation, transfer the fragments into a 15 ml centrifuge tube;

[0055] Sequentially use 0.1-0.5% trypsin (30 min) and 0.1-0.5% collagenase P (2 h) at 37°C with a volume fraction of 5% CO 2 digestion in an incubator;

[0056] After digestion, filter through a 100-mesh steel mesh, centrifuge at 1000 r / min for 5 min, and discard the supernatant. Wash twice, resuspend cells and count;

[0057] Take 5×10 4 A nucleated cell was plated in a T75 ...

Embodiment 2

[0062] The preparation method of embodiment 2 platelet-rich plasma is:

[0063] Under sterile conditions, collect 20ml of peripheral blood using sodium citrate anticoagulant and transfer it to a 50ml centrifuge tube;

[0064] Centrifuge at 1500rpm / min for 10min, transfer the supernatant to another 50ml centrifuge tube;

[0065] Centrifuge the supernatant obtained in the above step at 2000rpm / min for 10min, discard the supernatant, and leave 2ml of plasma to accommodate and dilute platelets;

[0066] Add 10ml of normal saline to the erythrocyte precipitation tube, mix well, centrifuge at 1500rpm / min for 10min, and transfer the supernatant to another 50ml centrifuge tube;

[0067] Centrifuge the supernatant obtained in the above step at 2000rpm / min for 10min, discard the supernatant, and collect the precipitate;

[0068] The platelet-plasma mixture obtained above is also combined with the platelet precipitate to obtain high-concentration platelet-rich plasma.

Embodiment 3

[0069] Example 3 Characteristics of synovial mesenchymal stem cells:

[0070] Immunophenotyping CD14, CD34, CD44, CD45, CD90, CD105, CD166, HLA-DR:

[0071] The mesenchymal stem cells derived from P4 human synovium were inoculated into T175 flasks;

[0072] When the cells reach 80-90% confluence, digest with 3ml of 0.025% trypsin diluent, transfer the cell suspension into a 50ml centrifuge tube and centrifuge at 1200rpm for 5min, discard the supernatant, and wash twice with normal saline;

[0073] Take 1×10 6 Cells / tubes are divided into 12 tubes, the first tube is a blank control (only containing mesenchymal stem cells), which is used to adjust the voltage of the flow cytometer, and the three tubes are respectively added with isotype control antibodies (PE-MOUSEIgG1, FITC-MOUSEIgG1, APC-MouseIgG1κ) 5 μl, add 5 μl of other corresponding antibodies to the remaining 8 tubes, mix well, and incubate at room temperature for 15 minutes in the dark;

[0074] Wash once with normal ...

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Abstract

The invention relates to the technical field of cell preparations, in particular to a synovial mesenchymal stem cell and PRP combined preparation for repairing articular cartilage injury as well as apreparation method and application thereof. The synovial mesenchymal stem cell and PRP combined preparation comprises synovial mesenchymal stem cells and PRP. The synovial mesenchymal stem cell and PRP combined preparation provided by the invention can effectively repair the articular cartilage injury, and the effect is better than the treatment effect of combination of sodium hyaluronate and umbilical cord mesenchymal stem cells.

Description

technical field [0001] The invention relates to the technical field of cell preparations, in particular to a synovial mesenchymal stem cell combined PRP preparation and its preparation method and application. In particular, it relates to the preparation of synovial mesenchymal stem cells combined with platelet-rich plasma and its preparation method for repairing articular cartilage damage. Mesenchymal stem cell preparation and its preparation and application fields. Background technique [0002] Articular cartilage is a viscoelastic substance, and the surface facing the joint cavity is very smooth, which facilitates the movement between bones. Cartilage itself has elasticity, so it can buffer the vibration and impact of connected bones during walking, jumping and other sports and reduce friction on the joint surface. Articular cartilage is arranged by sparse cells and has neither nerves nor blood vessels. Its nutrition is mainly supplied by synovial fluid and arterial bran...

Claims

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Application Information

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IPC IPC(8): A61K35/28C12N5/0775A61P19/04A61K35/16
CPCA61K35/28A61K35/16C12N5/0668A61P19/04C12N2509/00A61K2300/00
Inventor 刘家权牛振宇杨磊
Owner 国大生命科学产业集团(深圳)有限公司
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