Experimental method for enzymolysis, digestion and acquisition in vitro of rabbit knee cartilage unit

Abstract

The invention belongs to the technical field of the construction in vitro of tissue engineered cartilage and in particular relates to an experimental method for anzymolysis, digestion and acquisition in vitro of a rabbit knee cartilage unit. The experimental method comprises the following steps of: taking a rabbit knee joint, repeatedly shearing joint cartilages into fragment tissues, washing the fragment tissues, removing supernate and putting the fragment tissues in an aseptic cone bottle for later use; adding dispase enzyme and II type collagenase into the cone bottle in the proportion of 12ml DMEM-F12 culture solution per gram of cartilages, stirring the mixture and digesting to form digesting suspension; and filtering and centrifugating the digesting suspension and adding primary chondrocyte culture solution into the obtained product to prepare aseptic cartilage unit suspension. The experimental method has the advantages that: the acquisition method is simple and the repetitiveness is high; the experimental method more accords with the growing environment and the biological characteristics of the chondrocyte in vitro; and compared with the pure cell, the cartilage unit has obviously improved biomechanics characteristics and the defect of poor biomechanics characteristics of the chondrocyte serving as a seeded cell is overcome.

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 obtaining cartilage units by in vitro enzymolysis and digestion with rabbit knee articular cartilage as an experimental object. 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 subcul...

AI Technical Summary

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