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Cartilage graft and construction method thereof

A technology of cartilage transplantation and grafting, which is applied in the fields of medicine and biomedical engineering, and can solve problems such as limited donor sources, insufficient number of cells to regenerate large volumes of cartilage, loss of regenerated cartilage by high-generation chondrocytes, etc.

Pending Publication Date: 2018-07-31
SHANGHAI RESTHETIC BIO CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But it still has the following problems: 1. Insufficient number of low-generation chondrocytes: when the chondrocyte-material composite regenerates cartilage, the cell concentration is very high (>50×10 6 / ml)
Therefore, when using low-generation chondrocytes, the number of cells cannot meet the needs of regenerating large volumes of cartilage
However, in order to meet the cell number requirement, a large volume of cartilage tissue needs to be cut, and the source of donors is limited and traumatic
2. High-generation chondrocytes are easy to dedifferentiate: After chondrocytes are expanded in vitro, although the number of cells can meet the requirements, the high-generation chondrocytes are prone to dedifferentiation and lose the ability to regenerate cartilage
3. Difficulty in shaping: the chondrocyte-injectable material composite has the characteristics of easy flow, which makes it difficult to shape the composite after in vivo injection
4. The effect of cartilage regeneration is uncontrollable: Due to the influence of a series of factors such as cell viability, material degradation rate, and individual inflammatory response to materials, the quality and volume of the final regenerated cartilage cannot be precisely controlled
These problems greatly limit the wide application of cartilage regeneration technology in clinical repair of various cartilage defects.

Method used

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  • Cartilage graft and construction method thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0058] The preparation method of the cartilage graft provided by the invention comprises steps:

[0059] The first step is the acquisition and expansion of primary cells, that is, planar culture and expansion of chondrogenic cells, and passage to 1-8 generations; preferably the 1st, 2nd, 3rd, 4th, or 5th generation; more The best place is the 2nd generation - 5th generation;

[0060] The second step is the preparation of the perichondrium sheet, which is to inoculate the above-mentioned subcultured and amplified cells in an ordinary culture dish, and place them in the cartilage induction culture medium to form the perichondrium sheet by culturing in vitro;

[0061] In the third step, the cartilage graft is formed.

[0062] The method of planar culture expansion in the first step is, for example but not limited to, resuspending the isolated chondrocytes in high-sugar DMEM culture medium, then taking the cell suspension and inoculating it on a culture dish, culturing it under s...

Embodiment 1

[0086] Acquisition and expansion of primary cells

[0087] Take auricular cartilage with a size of about 1.0cm x 1.0cm from each animal, cut the cartilage pieces into pieces, add 0.25% trypsin (Hyclone Company, USA), shake and digest for 30min, then rinse with PBS 3 times, add 0.25% type II collagen Enzyme (Worthington Company, USA), shake and digest for 7-8h. The digested solution obtained was filtered with a 100-micron cell strainer, centrifuged, and the supernatant was removed. The precipitated cells were resuspended with high-sugar Dulbecco's modified Eagle's medium (DMEM, Gibco, USA), and the resulting cells were washed at 1×10 4 / cm2 concentration inoculated on 100mm Petri dishes at 37°C, 5% CO 2 , under saturated humidity conditions, culture and expand with DMEM medium (chondrocyte culture medium) containing 10% fetal bovine serum (U.S. Hyclone Company), 2ng / mlbFGF, 1% penicillin and streptomycin, until the chondrocytes grow to 70 Subculture when %-80% confluent.

Embodiment 2

[0089] Preparation of perichondrial slices 1

[0090] The P4 chondrocytes obtained in Example 1 were digested, centrifuged, resuspended, and mixed with 1×10 6 / cm 2 Concentrations were inoculated on ordinary culture dishes, and chondrogenic induction medium (1-50ng / ml insulin, 1-50mg / ml bFGF, 1-25ng / ml transferrin, 1-20mmol / ml IBMX, 1-50ng / ml sub Sodium selenate, 5×10 -2 -1mg / ml proline, 1-100mg / ml serum albumin, 1-20mg / ml β-mercaptoethanol, 1-50mg / ml L-glutamine, 1-20μg / ml linoleic acid and 10-200ng / ml ml ascorbic acid phosphate, 10ng / ml dexamethasone, 5ng / m TGFβ1 and 5ng / ml IGF) were cultured, and the medium was changed daily. After 4 weeks of in vitro culture, a 100mm-diameter perichondrial patch 1 can be uncovered in a petri dish for systematic pre-implantation evaluation and testing, such as: volume wet weight measurement, histological staining, collagen quantification, sterility testing, etc. . It was found that the wet weight of the perichondrium sheet was smaller,...

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Abstract

The invention discloses a cartilage graft and a construction method thereof. The cartilage graft is a cartilage membrane or injectable cartilage graft and contains a cartilage membrane or cartilage micro-tissue formed by in-vitro culture of cartilage cells.

Description

technical field [0001] The invention relates to the fields of medicine and biomedical engineering, and more particularly relates to a cartilage graft and a construction method thereof. Background technique [0002] Cartilage defect is a common clinical disease, which is mostly seen in the deformity and absence of ear and nasal cartilage caused by congenital reasons and trauma, cartilage defect caused by joint trauma and degeneration, etc. Due to the poor self-repair ability of cartilage tissue, the repair of various cartilage injuries has always been a difficult problem in clinical treatment. In recent years, minimally invasive surgery has developed rapidly, and injection filling therapy has become a research hotspot. Currently commonly used injectable filling materials mainly include hyaluronic acid, synthetic or animal-derived collagen and other degradable materials. Although the short-term effect of these injectable materials is satisfactory, the curative effect can onl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077A61F2/28
CPCA61F2/28A61F2002/2835C12N5/0655C12N2501/105C12N2501/15C12N2501/30
Inventor 曹谊林周广东李丹殷宗琦刘豫
Owner SHANGHAI RESTHETIC BIO CO LTD
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