The Effects of Transglutaminase on Perichondrocyte in Alginate Culture

Park, Jung Sun; Kong, Chang Bae; Kim, Choong Hyun; Ahn, Hyun Jeong; Jung, Mi Hyun; Seong, Sang Cheol; Kim, Hee Jung; Jo, Hyun Chul; Lee, Sang Hoon; Lee, Myung Chul

Abstract

PURPOSE
To investigate the effects of transglutaminase in the environment of extracellular matrix on perichondrocyte in alginate culture.
MATERIALS AND METHODS
Perichondrocyte cells were isolated from articular cartilage of New Zealand white rabbits by enzymatic digestion and maintained in monolayer culture. After 7 days, the cells were trypsinized and cultured in an alginate bead system. Four groups of the alginate beads were prepared as follow: containing 1 mg/ml of transglutaminase, 10 microgram/ml of fibronectin, mixture of 1mg/ml of TGase and 10 microgram/ml of fibronectin and only perichondrocytes as a control group. Cell proliferation was measure by [Methyl-3H] Thymidine uptake, and proteoglycan synthesis was measure by [35S] Sulfate uptake. The gene expression of integrin-alpha5, integrin-beta1 and type II collagen was analyzed by reverse transcription-polymerase chain reaction. Safranin-O staining was utilized for histological assessment of proteoglycan in extracellular matrix. One-way ANOVA was used to analyze the results statistically.
RESULTS
Mixture of transglutaminase and fibronectin exhibited high synthesis rates of proteoglycan and active cell proliferation compared with other groups. The gene expression of type II collagen did not show significant difference between groups. The gene expression of integrin-alpha5 was down-regulated in all groups with time. The gene expression of integrin-beta1 was not down-regulated with time only in mixture of transglutaminase and fibronectin. Histological staining of the secretions by Safranin-O staining was in agreement with the data of proteoglycan synthesis, and Safranin-O staining showed that more cell-to-cell aggregates is developed in the mixture of transglutaminase and fibronectin.
CONCLUSION
Mixture of transglutaminase and fibronectin can stimulate chondrocyte proliferation and proteoglycan synthesis, and integrin seems to modulate such interactions.