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Effect of type II collagen on biological characteristics of human chondrocytes

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Jiang Ping, Wei Wei, Zhao Mingcai, Chen Qiong, Wang Wei (Department of Orthopaedics, Affiliated Hospital of North Sichuan Medical College, Nanchong 637000, China)
 
Jiang Ping, female, born in 1983, Nanchong City, Sichuan Province, Han nationality, graduated from North Sichuan Medical College in 2012, master's degree, physician, mainly engaged in bone and joint injury repair research.
 
Corresponding author: Wei Peng, chief physician, Department of Orthopedics North Sichuan Medical College Hospital, Nanchong City, Sichuan Province 637000
 
Article highlights:
 
1. Collagen as a natural polymer biomaterial has been proven to be a scaffold for cartilage tissue engineering, and it can be used as a substrate to stimulate chondrocyte growth and can be used for in vitro chondrocyte culture, but for different types of collagen to stimulate cartilage. The ability is still controversial.
 
2. Experimentally, human chondrocytes were cultured simultaneously using type I and type II collagen-coated plates and common plates to study the effects of type I and type II collagen on the biological characteristics of human chondrocytes. It was found that collagen-coated plate cultured chondrocytes were superior to ordinary culture plates. The type II collagen-coated plates were more able to maintain cell morphology when cultured chondrocytes, prolong the time of dedifferentiation, and facilitate cell re-differentiation.
 
Key words:
 
Biomaterial; cartilage biomaterial; type I collagen culture plate; type II collagen culture plate; human chondrocyte; in vitro culture
 
Key words:
 
Collagen type I; collagen type II; chondrocytes
 
Fund funding:
 
Sichuan Provincial Health Department Project
 
Summary
 
BACKGROUND: Experiments have shown that collagen substrates have the effect of stimulating cartilage, but the ability to stimulate cartilage by different types of collagen remains controversial.
 
OBJECTIVE: To observe the effects of type I and type II collagen on the biological characteristics of human chondrocytes cultured in vitro.
 
METHODS: P3 human chondrocytes were added to common culture plates, type I collagen coated plates, and type II collagen coated plates to continue culture. After 10 days of culture, the cell growth curve was drawn by MTT method. After 28 days of culture, the secretion of collagen I and type II collagen from chondrocytes in three culture plates was detected by ELISA, polymerase chain reaction and dimethylmethylene blue colorimetric assay. The amount of protein and glycosaminoglycans.
 
RESULTS AND CONCLUSION: The number of chondrocytes in the type II collagen-coated plates was the highest, and the proliferation rate was 2 times that of the type I collagen-coated plates.
 
5 times the normal culture plate. The type II collagen secreted by chondrocytes in type II collagen-coated plates was the least, which was significantly different from that of common culture plates (P < 0.01). There was no significant difference between the results of type I collagen-coated plates. Sexual significance; type II collagen and glycosaminoglycan secreted by chondrocytes in type II collagen-coated plates, and the difference between the two culture plates was significant (P < 0.01). It indicates that the cultured chondrocytes cultured on collagen coated plates are superior to the common culture plates. The type II collagen coated culture plates can maintain the cell morphology and prolong the time of dedifferentiation, which is more conducive to cell re-differentiation.
 
Jiang Ping,Wei Wei,Zhao Mingcai,Chen Qiong,Wang Wei.Effects of type I and II collagen on the biological characteristics of human chondrocytes[J].
2014, 18(30): 4845-4850.
 
Effect of type I or type II collagen on biological characteristics of human chondrocytes
 
Jiang Ping, Master, Physician, Department of Orthopedics, Affiliated Hospital of North Sichuan Medical College, Nanchong 637000, Sichuan Province, China
 
Corresponding author: Wei Peng, Chief physician, Department of Orthopedics, Affiliated Hospital of North Sichuan Medical College, Nanchong 637000, Sichuan Province, China
 
Accepted: 2014-05-21
 
Jiang Ping, Wei Peng, Zhao Ming-cai, Chen Qiong, Wang Zi (Department of Orthopedics, Affiliated Hospital of North Sichuan Medical College, Nanchong 637000, Sichuan Province, China)
 
Abstract
 
BACKGROUND: Experiments have shown that the collagen substrate has the capability of stimulating cartilage generation, but the stimulating role of different types of collagen substrates remains controversial.
 
OBJECTIVE: To investigate the effect of type I and type II collagen on the biological characteristics of human chondrocytes cultured in vitro.
 
METHODS: Human chondrocytes at passage were cultured onto the ordinary culture plates (ordinary plate), type I collagen-coated culture plates (type I plate), and type II collagen-coated culture plates (type II plate). Cell growth curves were determined By MTT method after cells were cultured for 10 days. By ELISA, PCR, and 1,9-dimethyl methyleneblue technology, type I and type II collagen and glycosaminoglycan contents were quantitatively detected in cartilage cells 28 days after culture.
 
RESULTS AND CONCLUSION: The number of cartilage cells was the highest in type II plate, which was twice of that in type I plate and five times of that in ordinary plate. Cartilage cells in type II plate secreted the least amount of type I collagen, Which showed significant differences compared with the ordinary plate (P < 0.01) and had no statistically significant difference with type I plate (P > 0.01). Cartilage cells in type II plate secreted the most amount of type II collagen and glycosaminoglycan, The cartilage cells cultured in collagen plates are better than that cultured in ordinary culture plate, type II collagen culture plate is better than type I collagen culture plate in maintaining cell shape, extending the dedifferentiation Pattern, and promoting cell differentiation.
 
Subject headings: collagen type I; collagen type II; chondrocytes
 
Funding: a grant by Sichuan Provincial Health Ministry
 
Jiang P, Wei P, Zhao MC, Chen Q, Wang Z. Effect of type I or type II collagen on biological characteristics of human chondrocytes. Zhongguo Zuzhi Gongcheng Yanjiu. 2014;18(30):4845-4850.
 
introduction
 
With the development of tissue engineering, cartilage tissue engineering provides a more ideal method for repairing articular cartilage defects. How to obtain large-scale seed cells with proliferative activity is one of the biggest limiting factors currently facing.
 
In 1994, Brittberg [1] cultured healthy chondrocytes in vitro, obtained a large number of purified chondrocytes, and then transplanted them to the articular cartilage defect site to treat articular cartilage defects, and initiated a new technique for autologous chondrocyte transplantation for cartilage injury. This technology has been widely used in European countries after being officially approved by the US FDA in 1997. It is the key to the technology to obtain a large number of purified and regenerative chondrocytes. However, in the course of the experiment, it is found that the chondrocytes undergo dedifferentiation in vitro. Loss of cartilage ability.
 
In order to solve the dedifferentiation phenomenon of chondrocytes cultured in vitro, more and more studies have been reported. As early as 1975, experiments have shown that collagen substrates have the effect of stimulating cartilage [2]. Collagen, also known as collagen, is a structural protein in vertebrates. It is very rich in human body, accounting for 80%-90% of animal collagen fiber solids, accounting for 25%-30% of total protein in the body. The main component of the outer matrix [3]. Because collagen is a natural biological resource with a wide range of sources and unique biocompatibility, degradability, low immunogenicity, and unique properties such as high tensile strength, hemostatic properties and cell growth, it is increasingly The more attention you attach. Because collagen has the functions of stopping bleeding, promoting tissue regeneration and function recovery, it is made into medical equipment such as sponge, silk thread and film for use in general surgery, oral and vascular surgery, etc., and also because of its tissue compatibility. Degradability is used as a skin graft material for the repair of burns and wounds. In the 1880s, Zyderm collagen implants were used clinically as an injectable collagen preparation and have been used for nearly 30 years in the treatment of facial soft tissue deformation [4]. People have successfully made collagen into a variety of tissue engineering scaffolds, such as skin, bone tissue, trachea and vascular scaffolds, Award et al [5] mixed stem cells of autologous mesenchymal cells and collagen gel to make tendons Used for post-mortem repair.
 
Wakitani et al [6] repairs cartilage defects with collagen gel embedded in chondrocytes
 
trap. Collagen as a natural polymer biomaterial has been proven to be a scaffold for cartilage tissue engineering, and it acts as a substrate to stimulate chondrocyte growth and can be used for in vitro chondrocyte culture, but the ability to stimulate cartilage by different types of collagen remains there is controversy. In this experiment, human chondrocytes were cultured simultaneously using type I and type II collagen-coated plates and common plates.
 
Effect of type I and type II collagen on the biological characteristics of human chondrocytes.
 
Materials and Method
 
Design: Prospective experiment.
 
Time and place: From January 2008 to April 2009, he was completed at the Institute of Rheumatology and Immunology, Affiliated Hospital of North Sichuan Medical College.
 
MATERIALS: Collection of orthopedics in the Department of Orthopaedics, Affiliated Hospital of North Sichuan Medical College
Femoral head specimens of 20 patients with total hip replacement, aged 56-84 years, mean 66 years. The donor gave informed consent to the experiment and signed an informed consent form.
 
experimental method:
 
Isolation and culture of chondrocytes: After collecting the collected articular cartilage with a volume fraction of 75% ethanol, the soft tissue of the joint surface and the synovial membrane that may be covered thereon are scraped off with a surgical blade, and the thin layer of the cartilage surface of the joint surface is scraped off. Make the cut cartilage tissue as thin as possible and not cut into the subchondral bone. The collected cartilage tissue pieces were rinsed three times in PBS containing penicillin (100 U/mL) and streptomycin (100 U/mL) to wash away the blood on the surface of the cartilage.