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Preparation method of exosome-containing cartilage repair material

A cartilage repair and exosome technology, which is applied in the preparation of cartilage repair materials and in the field of cartilage repair materials, can solve the problems of difficult preservation, no repair of blood vessels, and easy inactivation of growth factors, etc., to achieve easy access, good guidance for tissue formation, The effect of high bionicity

Inactive Publication Date: 2020-08-07
百澳瑞派(天津)生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The constructed cartilage material is not only similar in composition to natural cartilage, but also its three-dimensional configuration can satisfy the three-dimensional place for growth, proliferation and migration, and provide enough space for cells to grow. However, the existing collagen-based bone materials can only promote osteogenesis. Cell proliferation has no repair effect on blood vessels, and the added growth factors are also easily inactivated and difficult to preserve

Method used

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  • Preparation method of exosome-containing cartilage repair material

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Experimental program
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Effect test

Embodiment 1

[0033] (1) Preparation steps of exosomes:

[0034] Step 1: Construct a plasmid for overexpression of miRNA140 by means of genetic engineering.

[0035] Based on the pCDH-CMV-MCS-EF1-copGFP lentiviral expression vector, the miRNA-140 gene was integrated to obtain the miRNA-140 overexpression lentiviral vector pCDH-CMV-MCS-EF1-copGFP-140.

[0036] Step 2: Pack and obtain lentivirus, and transfect 293T.

[0037] Use lentiviral packaging plasmids pSPAX2 and pMD2.G to package pCDH-CMV-MCS-EF1-copGFP-140, transfect 293T cells, and enrich high-titer virus liquid.

[0038] Step 3: infect bone marrow mesenchymal stem cells.

[0039] The packaged virus was used to infect bone marrow mesenchymal stem cells, and bone marrow mesenchymal stem cells hBMMSCs-140 overexpressing miRNA-140 were obtained.

[0040] Step 4: Cultivate hBMMSC-140, isolate exosome Exso-140 overexpressing miRNA-140 from the culture medium, add a preservation solution containing sodium gluconate and inorganic salts, ...

Embodiment 2

[0045] (1) Preparation steps of exosomes

[0046] Step 1: Construct a plasmid for overexpression of miRNA140 by means of genetic engineering.

[0047] Based on the pCDH-EF1-Luc2-T2A-tdTomato lentiviral expression vector, the miRNA-140 gene was integrated to obtain the miRNA-140 overexpression lentiviral vector pCDH-EF1-Luc2-T2A-tdTomato-140.

[0048] Step 2: Pack and obtain lentivirus, and transfect 293T.

[0049] Use lentiviral packaging plasmids pSPAX2 and pMD2.G to package pCDH-EF1-Luc2-T2A-tdTomato-140, transfect 293T cells, and enrich high-titer virus liquid.

[0050] Step 3: infect bone marrow mesenchymal stem cells.

[0051] The packaged virus was used to infect bone marrow mesenchymal stem cells, and bone marrow mesenchymal stem cells hBMMSCs-140 overexpressing miRNA-140 were obtained.

[0052] Step 4: Cultivate hBMMSC-140, isolate exosome Exso-140 overexpressing miRNA-140 from the culture medium, add a preservation solution containing sodium gluconate and inorganic...

Embodiment 3

[0057] Preparation steps of exosomes

[0058] Step 1: Construct a plasmid for overexpression of miRNA140 by means of genetic engineering.

[0059] Based on the pCDF1-MCS2-EF1-copGFP lentiviral expression vector, the miRNA-140 gene was integrated to obtain the miRNA-140 overexpression lentiviral vector pCDF1-MCS2-EF1-copGFP-140.

[0060] Step 2: Pack and obtain lentivirus, and transfect 293T.

[0061] Use lentiviral packaging plasmids pSPAX2 and pMD2.G to package pCDF1-MCS2-EF1-copGFP-140, transfect 293T cells, and enrich high-titer virus liquid.

[0062] Step 3: infect bone marrow mesenchymal stem cells.

[0063] The packaged virus was used to infect bone marrow mesenchymal stem cells, and bone marrow mesenchymal stem cells hBMMSCs-140 overexpressing miRNA-140 were obtained.

[0064] Step 4: Cultivate hBMMSC-140, isolate exosome Exso-140 overexpressing miRNA-140 from the culture medium, add a preservation solution containing sodium gluconate and inorganic salts, and store a...

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Abstract

The invention relates to a preparation method of an exosome-containing cartilage repair material. The method comprises the following steps of exosome preparation, scaffold material preparation and composite material preparation. The exosome preparation comprises the following steps: step 1, constructing miRNA140 overexpressed plasmids by genetic engineering means; step 2, performing packaging to obtain lentiviruses, and transfecting 293T; step 3, infecting mesenchymal stem cells; and step 4, culturing hBMMSC-140, separating the exosome Exso-140 for over-expressed miRNA-140 from a culture medium, adding a preservation solution containing sodium gluconate and inorganic salt, and performing preserving at a temperature of 80 DEG C below zero. The invention aims at preparing a bionic cartilagescaffold material taking bi-component collagen and hydroxyapatite as elements. The exosome is effectively embedded in the bionic bone material, the original activity of the exosome is maintained, andthe functional cartilage repair material with activity is formed; and the material can play a role in osteoblast proliferation, can repair vascular injury, and enhances the functionality of the bionicbone material.

Description

technical field [0001] The invention belongs to the field of tissue engineering and relates to cartilage repair material technology, in particular to a preparation method of cartilage repair material containing exosomes. Background technique [0002] The surface of articular cartilage is smooth and elastic, which can absorb and buffer stress to the greatest extent, reduce the friction between two adjacent bones, and buffer the vibration generated during exercise. However, articular cartilage damage is difficult to heal itself. The current treatment methods mainly include cartilage transplantation and joint prosthesis replacement with metal materials or polymer materials. However, both methods have their own problems in clinical application, such as limited sources and loosening after replacement. [0003] In recent years, with the development of cell biology and biomaterial science, tissue engineering has emerged as the times require. It uses the principles and methods of li...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61L27/12A61L27/24A61L27/38A61L27/50A61L27/56C12N15/867C12N5/10
CPCA61L27/12A61L27/24A61L27/3834A61L27/3895A61L27/50A61L27/56A61L2430/06C12N5/0663C12N15/86C12N2510/00C12N2533/54C12N2740/15043
Inventor 郭雅楠柏玮李志宏蒋彩云
Owner 百澳瑞派(天津)生物科技有限公司
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