Effects of IGF (Insulin-like growth factor) on sub-populated articular chondrocytes by Percoll density gradient.
- Author:
Hyun Jung LEE
1
;
Hyun Ju KIM
;
Hong Sik CHO
;
So Ra PARK
;
Byoung Hyun MIN
Author Information
1. Department of Biomedical Engineering, Ajou University, Suwon, Korea. bhmin@ajou.ac.kr
- Publication Type:Original Article
- Keywords:
Articular chondrocyte;
Percoll density gradient;
IGF (insulin-like growth factor)
- MeSH:
Aggrecans;
Anabolic Agents;
Blotting, Western;
Cartilage;
Cartilage, Articular;
Chondrocytes*;
Collagen;
Collagen Type II;
Collagenases;
Cytokines;
Digestion;
Extracellular Matrix;
Hand;
Humans;
Immunohistochemistry;
Intercellular Signaling Peptides and Proteins;
Knee;
Male;
Pentobarbital;
Population Characteristics;
Proteoglycans;
Rabbits;
Tissue Inhibitor of Metalloproteinase-2
- From:Journal of Korean Orthopaedic Research Society
2004;7(1):49-59
- CountryRepublic of Korea
- Language:Korean
-
Abstract:
PURPOSE: Articular chondrocytes have been known to have heterogeneity in articular cartilage. The different responses of chondrocytes to various cytokines and growth factors have been reported. These variations are likely a result of metabolic differences among the cell populations. We used the Percoll density gradient method to separate chondrocytes from articular cartilage into distinct subpopulations. Several growth factors are known to enhance the synthesis of cartilage matrix. In particular, IGF has specific anabolic effects. Addition of IGF to chondrocytes increased the synthesis of proteoglycans and collagen type-II while inhibiting the degradation and release of proteoglycans. MATERIALS AND METHODS: Chondrocytes were isolated from rabbit knee articular cartilage by collagenase digestion. In brief, male rabbits weighing 250g were euthanized by injecting an overdose of Nembutal, and nonfibrillated articular cartilage of the knee was removed by sterile dissection. Isotonic Percoll was mixed with 10x PBS to give a 60% stock solution. This was further diluted with PBS to give Percoll concentrations of 10, 20, 30, 40, 50, and 60%. RT-PCR, western blot analysis, immunocytochemistry, and immunohistochemistry were done for examination of collagen type II and aggrecan as the specific marker of extracellular matrix and proteoglycan synthesis on cultured chondrocytes. RESULTS: The sub-populated cells were proliferated variously. On the other hand, the addition of IGF to the sub-populated cells increased the proliferation in all fractions. Also the expression of collagen type-II and TIMP-2 was increased by IGF treatment. After alginate culture, collagen type-II expression was not significantly different between the IGF treated and the control groups in high density fractions. However, the addition of IGF to chondrocytes increased the expression of collagen type-II in low density fractions. The expression of collagen type-II after IGF addition was decreased in monolayer culture while it was increased in alginate culture. CONCLUSION: The effects of IGF are various among the subpopulated chondrocytes. These results will provide useful information for the separation of articular chondrocytes with an active metabolic activity and extracellular matrix for the investigation of the pathogenesis of articular cartilage.
