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Experimental method for establishing tissue engineering cartilage in vitro by taking knee-joint cartilage unit of rabbit as seed cell

A seed cell and tissue engineering technology, applied in prosthetics, medical science, etc., can solve the problems of failure to form hyaline cartilage tissue, decrease in viscoelasticity, and restrict the development of tissue engineering technology, and achieve the effect of simple acquisition method and good repeatability

Inactive Publication Date: 2013-04-03
THE SECOND HOSPITAL OF SHANXI MEDICAL UNIV +1
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Problems solved by technology

However, in the process of in vitro culture and subculture of chondrocytes, the viscoelasticity gradually decreases, the fragility increases, and the function of chondrocytes degenerates relatively quickly. During the process of repairing cartilage defects in vivo, hyaline cartilage tissue that conforms to the characteristics of normal cartilage cannot be formed, which has restricted the development of tissue engineering technology. one of the biggest obstacles

Method used

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Embodiment Construction

[0021] Concrete steps of the present invention are as follows:

[0022] (1) Rabbit knee joints were collected under aseptic conditions. The knee joint was opened in an ultra-clean workbench, and the full-thickness cartilage of the femur and tibial plateau of the knee joint was scraped with a sharp knife. The articular cartilage was repeatedly cut into pieces with small scissors, rinsed with sterile D-Hanks solution, the supernatant was discarded, weighed, and placed in a sterile Erlenmeyer flask for later use.

[0023] (2) Add 0.3% dispase (a neutral lyase, Sigma, USA) and 0.2% type II collagenase (Sigma, USA) to the Erlenmeyer flask at the rate of 12 mL DMEM-F12 culture solution / g cartilage. Both enzymes were dissolved in sterile DMEM-F12 culture solution (HyClone, USA), filtered through a 0.22 μm disposable filter, and sterilized. Place the Erlenmeyer flask with culture solution at 37°C, 5% CO 2 On the magnetic stirrer in the incubator, stir at a slow speed, and carry out...

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Abstract

The invention belongs to the technical field of in-vitro establishment of tissue engineering cartilages, more particularly to an experimental method for establishing tissue engineering cartilage in vitro by taking a knee-joint cartilage unit of a rabbit as a seed cell. The experimental method comprises the following steps of: taking a keen joint of a rabbit, repeatedly cutting the joint cartilage into fragmentized tissues, washing, removing supernatant away and putting in a sterile conical flask for later use; adding dispase and II-type collagenase into the conical flask according to 12mL DMEM-F12 culture solution / g cartilage, stirring and digesting to form a digested suspension solution; filtering and centrifuging the digested suspension solution, adding a primary chondrocyte culture solution to prepare a sterile chondrocyte suspension solution, and carrying out sodium alginate gel three-dimensional culture on the obtained cartilage unit to construct the tissue engineering cartilage. The invention has the beneficial effects that: the cartilage unit has stable form and multiplication situation in the in-vitro long-term culturing process, the physiological function of substrate ingredients, such as secreted II-type collagen, and the like is improved, and the effect of repairing cartilage injury by adopting a tissue engineering method is expectedly accelerated.

Description

technical field [0001] The invention belongs to the technical field of in vitro construction of tissue engineered cartilage, and specifically relates to an experimental method for constructing tissue engineered cartilage in vitro by using rabbit knee articular cartilage units as seed cells. Background technique [0002] During the in vitro construction of tissue-engineered cartilage, the selection of ideal seed cells is the most critical and difficult point in current research. At present, in domestic and foreign literature reports, the most commonly used seed cells for articular cartilage tissue engineering are chondrocytes. The conventional enzymatic digestion method in vitro is: 0.4% Pronase enzyme is used to digest 12mL DMEM-F12 culture solution / g cartilage at 37°C for 90 minutes. After replacing the culture solution, 0.025% type II collagenase is digested overnight to obtain knee joint chondrocytes. However, in the process of in vitro culture and subculture of chondroc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61L27/38A61L27/20
Inventor 卫小春段王平孙振伟李琦
Owner THE SECOND HOSPITAL OF SHANXI MEDICAL UNIV
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