J Korean Orthop Res Soc.  2004 Apr;7(1):49-59.

Effects of IGF (Insulin-like growth factor) on sub-populated articular chondrocytes by Percoll density gradient

• Lee HJ
• Kim HJ
• Cho HS
• Park SR
• Min BH

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.

Keyword

Articular chondrocyte; Percoll density gradient; IGF (insulin-like growth factor)

MeSH Terms

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
Aggrecans
Anabolic Agents
Collagen
Collagen Type II
Collagenases
Cytokines
Intercellular Signaling Peptides and Proteins
Pentobarbital
Proteoglycans
Tissue Inhibitor of Metalloproteinase-2